Modulation of apolipoprotein c-III expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of apolipoprotein C-III. The compositions comprise oligonucleotides, targeted to nucleic acid encoding apolipoprotein C-III. Methods of using these compounds for modulation of apolipoprotein C-III expression and for diagnosis and treatment of disease associated with expression of apolipoprotein C-III are provided.

RELATED APPLICATIONS

This application is the U.S. national phase application under 35 U.S.C.§371 of International Application No. PCT/US2004/010946, filed Apr. 15,2004, designating the United States and published in English on Nov. 4,2004 as WO2004/093783, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/418,780, filed Apr. 16, 2003 now U.S. Pat. No.7,598,227.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of apolipoprotein C-III. In particular, this inventionrelates to compounds, particularly oligonucleotide compounds, which, inpreferred embodiments, hybridize with nucleic acid molecules encodingapolipoprotein C-III. Such compounds are shown herein to modulate theexpression of apolipoprotein C-III.

BACKGROUND OF THE INVENTION

Lipoproteins are globular, micelle-like particles that consist of anon-polar core of acylglycerols and cholesteryl esters surrounded by anamphiphilic coating of protein, phospholipid and cholesterol.Lipoproteins have been classified into five broad categories on thebasis of their functional and physical properties: chylomicrons, whichtransport dietary lipids from intestine to tissues; very low densitylipoproteins (VLDL); intermediate density lipoproteins (IDL); lowdensity lipoproteins (LDL); all of which transport triacylglycerols andcholesterol from the liver to tissues; and high density lipoproteins(HDL), which transport endogenous cholesterol from tissues to the liver.

Lipoprotein particles undergo continuous metabolic processing and havevariable properties and compositions. Lipoprotein densities increasewithout decreasing particle diameter because the density of their outercoatings is less than that of the inner core. The protein components oflipoproteins are known as apolipoproteins. At least nine apolipoproteinsare distributed in significant amounts among the various humanlipoproteins.

Apolipoprotein C-III is a constituent of HDL and of triglyceride-richlipoproteins and has a role in hypertriglyceridemia, a risk factor forcoronary artery disease. Apolipoprotein C-III slows this clearance oftriglyceride-rich lipoproteins by inhibiting lipolysis, both throughinhibition of lipoprotein lipase and by interfering with lipoproteinbinding to the cell-surface glycosaminoglycan matrix (Shachter, Curr.Opin. Lipidol., 2001, 12, 297-304).

The gene encoding human apolipoprotein C-III (also called APOC3,APOC-III, APO CIII, and APO C-III) was cloned in 1984 by three researchgroups (Levy-Wilson et al., DNA, 1984, 3, 359-364; Protter et al., DNA,1984, 3, 449-456; Sharpe et al., Nucleic Acids Res., 1984, 12,3917-3932). The coding sequence is interrupted by three introns (Protteret al., DNA, 1984, 3, 449-456). The human apolipoprotein C-III gene islocated approximately 2.6 kB to the 3′ direction of the apolipoproteinA-1 gene and these two genes are convergently transcribed (Karathanasis,Proc. Natl. Acad. Sci. U.S.A., 1985, 82, 6374-6378). Also cloned was avariant of human apolipoprotein C-III with a Thr74 to Ala74 mutationfrom a patient with unusually high level of serum apolipoprotein C-III.As the Thr74 is O-glycosylated, the Ala74 mutant therefore resulted inincreased levels of serum apolipoprotein C-III lacking the carbohydratemoiety (Maeda et al., J. Lipid Res., 1987, 28, 1405-1409).

Five polymorphisms have been identified in the promoter region of thegene: C(−641) to A, G(−630) to A, T(−625) to deletion, C(−482) to T andT(−455) to C). All of these polymorphisms are in linkage disequilibriumwith the SstI polymorphism in the 3′ untranslated region. The SstI sitedistinguishes the S1 and S2 alleles and the S2 allele has beenassociated with elevated plasma triglyceride levels (Dammerman et al.,Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 4562-4566). The apolipoproteinC-III promoter is downregulated by insulin and this polymorphic siteabolishes the insulin regulation. Thus the potential overexpression ofapolipoprotein C-III resulting from the loss of insulin regulation maybe a contributing factor to the development of hypertriglyceridemiaassociated with the S2 allele (Li et al., J. Clin. Invest., 1995, 96,2601-2605). The T(−455) to C polymorphism has been associated with anincreased risk of coronary artery disease (Olivieri et al., J. LipidRes., 2002, 43, 1450-1457).

In addition to insulin, other regulators of apolipoprotein C-III geneexpression have been identified. A response element for the nuclearorphan receptor rev-erb alpha has been located at positions −23/−18 inthe apolipoprotein C-III promoter region and rev-erb alpha decreasesapolipoprotein C-III promoter activity (Raspe et al., J. Lipid Res.,2002, 43, 2172-2179). The apolipoprotein C-III promoter region −86 to−74 is recognized by two nuclear factors CIIIb1 and CIIIB2 (Ogami etal., J. Biol. Chem., 1991, 266, 9640-9646). Apolipoprotein C-IIIexpression is also upregulated by retinoids acting via the retinoid Xreceptor, and alterations in retinoid X receptor abundance affectsapolipoprotein C-III transcription (Vu-Dac et al., J. Clin. Invest.,1998, 102, 625-632). Specificity protein 1 (Sp1) and hepatocyte nuclearfactor-4 (HNF-4) have been shown to work synergistically totransactivate the apolipoprotein C-III promoter via the HNF-4 bindingsite (Kardassis et al., Biochemistry, 2002, 41, 1217-1228). HNF-4 alsoworks in conjunction with SMAD3-SMAD4 to transactivate theapolipoprotein C-III promoter (Kardassis et al., J. Biol. Chem., 2000,275, 41405-41414).

Transgenic and knockout mice have further defined the role ofapolipoprotein C-III in lipolysis. Overexpression of apolipoproteinC-III in transgenic mice leads to hypertriglyceridemia and impairedclearance of VLDL-triglycerides (de Silva et al., J. Biol. Chem., 1994,269, 2324-2335; Ito et al., Science, 1990, 249, 790-793). Knockout micewith a total absence of the apolipoprotein C-III protein exhibitedsignificantly reduced plasma cholesterol and triglyceride levelscompared with wild-type mice and were protected from postprandialhypertriglyceridemia (Maeda et al., J. Biol. Chem., 1994, 269,23610-23616).

Currently, there are no known therapeutic agents that affect thefunction of apolipoprotein C-III. The hypolipidemic effect of thefibrate class of drugs has been postulated to occur via a mechanismwhere peroxisome proliferator activated receptor (PPAR) mediates thedisplacement of HNF-4 from the apolipoprotein C-III promoter, resultingin transcriptional suppression of apolipoprotein C-III (Hertz et al., J.Biol. Chem., 1995, 270, 13470-13475). The statin class of hypolipidemicdrugs also lower triglyceride levels via an unknown mechanism, whichresults in increases in lipoprotein lipase mRNA and a decrease in plasmalevels of apolipoprotein C-III (Schoonjans et al., FEBS Lett., 1999,452, 160-164). Consequently, there remains a long felt need foradditional agents capable of effectively inhibiting apolipoprotein C-IIIfunction.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for modulatingapolipoprotein C-III expression. Antisense technology is emerging as aneffective means for reducing the expression of specific gene productsand is uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of apolipoprotein C-IIIexpression.

The present invention is directed to compounds, especially nucleic acidand nucleic acid-like oligomers, which are targeted to a nucleic acidencoding apolipoprotein C-III, and which modulate the expression ofapolipoprotein C-III. Pharmaceutical and other compositions comprisingthe compounds of the invention are also provided.

Further provided are methods of screening for modulators ofapolipoprotein C-III and methods of modulating the expression ofapolipoprotein C-III in cells, tissues or animals comprising contactingsaid cells, tissues or animals with one or more of the compounds orcompositions of the invention. In these methods, the cells or tissuesare contacted in vivo. Alternatively, the cells or tissues are contactedex vivo.

Methods of treating an animal, particularly a human, suspected of havingor being prone to a disease or condition associated with expression ofapolipoprotein C-III are also set forth herein. Such methods compriseadministering a therapeutically or prophylactically effective amount ofone or more of the compounds or compositions of the invention to theperson in need of treatment.

Also provided is a method of making a compound of the inventioncomprising specifically hybridizing in vitro a first nucleobase strandcomprising a sequence of at least 8 contiguous nucleobases of thesequence set forth in SEQ ID NO: 4 and/or SEQ ID NO: 18 to a secondnucleobase strand comprising a sequence sufficiently complementary tosaid first strand so as to permit stable hybridization.

The invention further provides a compound of the invention for use intherapy.

The invention further provides use of a compound or composition of theinvention in the manufacture of a medicament for the treatment of anyand all conditions disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION A. Overview of the Invention

The present invention employs compounds, preferably oligonucleotides andsimilar species for use in modulating the function or effect of nucleicacid molecules encoding apolipoprotein C-III. This is accomplished byproviding oligonucleotides that specifically hybridize with one or morenucleic acid molecules encoding apolipoprotein C-III.

As used herein, the terms “target nucleic acid” and “nucleic acidmolecule encoding apolipoprotein C-III” have been used for convenienceto include DNA encoding apolipoprotein C-III, RNA (including pre-mRNAand mRNA or portions thereof) transcribed from such DNA, and also cDNAderived from such RNA.

The hybridization of a compound of this invention with its targetnucleic acid is generally referred to as “antisense”. Consequently, themechanism included in the practice of some preferred embodiments of theinvention is referred to herein as “antisense inhibition.” Suchantisense inhibition is typically based upon hydrogen bonding-basedhybridization of oligonucleotide strands or segments such that at leastone strand or segment is cleaved, degraded, or otherwise renderedinoperable. In this regard, it is presently preferred to target specificnucleic acid molecules and their functions for such antisenseinhibition.

The functions of DNA to be interfered with include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includefunctions such as translocation of the RNA to a site of proteintranslation, translocation of the RNA to sites within the cell which aredistant from the site of RNA synthesis, translation of protein from theRNA, splicing of the RNA to yield one or more RNA species, and catalyticactivity or complex formation involving the RNA which may be engaged inor facilitated by the RNA. One preferred result of such interferencewith target nucleic acid function is modulation of the expression ofapolipoprotein C-III. In the context of the present invention,“modulation” and “modulation of expression” mean either an increase(stimulation) or a decrease (inhibition) in the amount or levels of anucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition isoften the preferred form of modulation of expression and mRNA is often apreferred target nucleic acid.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,the preferred mechanism of pairing involves hydrogen bonding, which maybe Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases (nucleobases) ofthe strands of oligomeric compounds. For example, adenine and thymineare complementary nucleobases, which pair through the formation ofhydrogen bonds. Hybridization can occur under varying circumstances.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand are different in different circumstances. In the context of thisinvention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleobases of an oligomeric compound. For example,if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound), is capable of hydrogen bonding with a nucleobaseat a certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position ofhydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases that can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termsthat are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

It is understood in the art that the sequence of the antisense compoundof this invention can be, but need not be, 100% complementary to that ofthe target nucleic acid to be specifically hybridizable. Moreover, anoligonucleotide may hybridize over one or more segments such thatintervening or adjacent segments are not involved in the hybridizationevent (e.g., a loop structure or hairpin structure). In one embodiment,the antisense compounds of the present invention comprise at least 70%,or at least 75%, or at least 80%, or at least 85% sequencecomplementarity to a target region within the target nucleic acid. Inanother embodiment, the antisense compounds of this invention comprise90% sequence complementarity and even more preferably comprise 95% or atleast 99% sequence complementarity to the target region within thetarget nucleic acid sequence to which they are targeted. Preferably, theantisense compounds comprise at least 8 contiguous nucleobases of anantisense compound disclosed herein. For example, an antisense compoundin which 18 of 20 nucleobases of the antisense compound arecomplementary to a target region, and would therefore specificallyhybridize, would represent 90 percent complementarity. In this example,the remaining noncomplementary nucleobases may be clustered orinterspersed with complementary nucleobases and need not be contiguousto each other or to complementary nucleobases. As such, an antisensecompound which is 18 nucleobases in length having 4 (four)noncomplementary nucleobases which are flanked by two regions ofcomplete complementarity with the target nucleic acid would have 77.8%overall complementarity with the target nucleic acid and would thus fallwithin the scope of the present invention. Percent complementarity of anantisense compound with a region of a target nucleic acid can bedetermined routinely using BLAST programs (basic local alignment searchtools) and PowerBLAST programs known in the art (Altschul et al., J.Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7,649-656).

Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Insome preferred embodiments, homology, sequence identity orcomplementarity, between the oligomeric and target is between about 50%to about 60%. In some embodiments, homology, sequence identity orcomplementarity, is between about 60% and about 70%. In preferredembodiments, homology, sequence identity or complementarity, is betweenabout 70% and about 80%. In more preferred embodiments, homology,sequence identity or complementarity, is between about 80% and about90%. In some preferred embodiments, homology, sequence identity orcomplementarity, is about 90%, about 92%, about 94%, about 95%, about96%, about 97%, about 98%, or about 99%.

B. Compounds of the Invention

According to the present invention, compounds include antisenseoligomeric compounds, antisense oligonucleotides, ribozymes, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds that hybridize to at least aportion of the target nucleic acid. As such, these compounds may beintroduced in the form of single-stranded, double-stranded, circular orhairpin oligomeric compounds and may contain structural elements such asinternal or terminal bulges or loops. Once introduced to a system, thecompounds of the invention may elicit the action of one or more enzymesor structural proteins to effect modification of the target nucleicacid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds which are“DNA-like” elicit RNAse H. Activation of RNAse H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNAseIII and ribonuclease L family of enzymes.

While the preferred form of antisense compound is a single-strandedantisense oligonucleotide, in many species the introduction ofdouble-stranded structures, such as double-stranded RNA (dsRNA)molecules, induces potent and specific antisense-mediated reduction ofthe function of a gene or its associated gene products. This phenomenonoccurs in both plants and animals and is believed to have anevolutionary connection to viral defense and transposon silencing.

The first evidence that dsRNA could lead to gene silencing in animalscame in 1995 from work in the nematode, Caenorhabditis elegans (Guo andKempheus, Cell, 1995, 81, 611-620). The primary interference effects ofdsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci.USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanismdefined in Caenorhabditis elegans resulting from exposure todouble-stranded RNA (dsRNA) has since been designated RNA interference(RNAi). This term has been generalized to mean antisense-mediated genesilencing involving the introduction of dsRNA leading to thesequence-specific reduction of endogenous targeted mRNA levels (Fire etal., Nature, 1998, 391, 806-811). Recently, the single-stranded RNAoligomers of antisense polarity of the dsRNAs have been reported to bepotent inducers of RNAi (Tijsterman et al., Science, 2002, 295,694-697).

In the context of this invention, the term “oligomeric compound” refersto a polymer or oligomer comprising a plurality of monomeric units. Inthe context of this invention, the term “oligonucleotide” refers to anoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA) or mimetics, chimeras, analogs and homologs thereof. This termincludes oligonucleotides composed of naturally occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

The oligonucleotides of the present invention also include modifiedoligonucleotides in which a different nucleobase is present at one ormore of the nucleotide positions in the oligonucleotide. For example, ifthe first nucleotide is adenosine, modified oligonucleotides may beproduced that contain thymidine, quanosine or cytidine at this position.This may be done at any of the positions of the oligonucleotide. Theseoligonucleotides are then tested using the methods described herein todetermine their ability to inhibit expression of apolipoprotein C-III.

While oligonucleotides are a preferred form of the compounds of thisinvention, the present invention comprehends other families of compoundsas well, including but not limited to oligonucleotide analogs andmimetics such as those described herein.

The compounds in accordance with this invention preferably comprise fromabout 8 to about 80 nucleobases (i.e. from about 8 to about 80 linkednucleosides). One of ordinary skill in the art will appreciate that theinvention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

In one preferred embodiment, the compounds of the invention are 12 to 50nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases inlength.

In another preferred embodiment, the compounds of the invention are 15to 30 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

Particularly preferred compounds are oligonucleotides from about 12 toabout 50 nucleobases, even more preferably those comprising from about15 to about 30 nucleobases.

Antisense compounds 8-80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

Exemplary preferred antisense compounds include oligonucleotidesequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains about 8to about 80 nucleobases). Similarly preferred antisense compounds arerepresented by oligonucleotide sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same oligonucleotide beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 8 to about 80 nucleobases).Exemplary compounds of this invention from a variety of mammaliansources, including human, may be found identified in the Examples andlisted in Tables 1 through 21. One having skill in the art armed withthe preferred antisense compounds illustrated herein will be able,without undue experimentation, to identify further preferred antisensecompounds.

C. Targets of the Invention

“Targeting” an antisense compound to a target nucleic acid moleculeencoding apolipoprotein C-III, in the context of this invention, can bea multi-step process. The process usually begins with the identificationof a target nucleic acid whose function is to be modulated. This targetnucleic acid may be, for example, a cellular gene (or mRNA transcribedfrom the gene) whose expression is associated with a particular disorderor disease state, or a nucleic acid molecule from an infectious agent.In the present invention, the target nucleic acid encodes apolipoproteinC-III.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes, having translation initiation codons withthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and5′-CUG, have been shown to function in vivo. Thus, the terms“translation initiation codon” and “start codon” can encompass manycodon sequences, even though the initiator amino acid in each instanceis typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA transcribed from a gene encodingapolipoprotein C-III, regardless of the sequence(s) of such codons. Itis also known in the art that a translation termination codon (or “stopcodon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAGand 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions that may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, apreferred region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsopreferred to target the 5′ cap region.

Accordingly, the present invention provides antisense compounds thattarget a portion of nucleobases 1-533 as set forth in SEQ ID NO: 18. Inone embodiment, the antisense compounds target at least an 8 nucleobaseportion of nucleobases 1-533 as set forth in SEQ ID NO: 18 and Tables 1and 4. In another embodiment, the antisense compounds target at least an8 nucleobase portion of nucleobases comprising the 5′ UTR as set forthin SEQ ID NO: 18 and Tables 1 and 4. In another embodiment, theantisense compounds target at least an 8 nucleobase portion ofnucleobases comprising the 3′ UTR as set forth in SEQ ID NO: 18 andTables 1 and 4. In another embodiment, the antisense compounds target atleast an 8 nucleobase portion of nucleobases comprising the codingregion as set forth in SEQ ID NO: 18 and Tables 1 and 4. In still otherembodiments, the antisense compounds target at least an 8 nucleobaseportion of a “preferred target segment” (as defined herein) as set forthin Table 3.

Further, the present invention provides antisense compounds that targeta portion of nucleobases 1-3958 as set forth in SEQ ID NO: 4. In oneembodiment, the antisense compounds target at least an 8 nucleobaseportion of nucleobases 1-3958 as set forth in SEQ ID NO: 4 and Tables 1and 4. In another embodiment, the antisense compounds target at least an8 nucleobase portion of nucleobases comprising the 5′ UTR as set forthin SEQ ID NO: 4 and Tables 1 and 4. In another embodiment, the antisensecompounds target at least an 8 nucleobase portion of nucleobasescomprising the 3′ UTR as set forth in SEQ ID NO: 4 and Tables 1 and 4.In another embodiment, the antisense compounds target at least an 8nucleobase portion of nucleobases comprising the coding region as setforth in SEQ ID NO: 4 and Tables 1 and 4. In still other embodiments,the antisense compounds target at least an 8 nucleobase portion of a“preferred target segment” (as defined herein) as set forth in Table 3.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also preferred target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesare known as “fusion transcripts”. It is also known that introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

Alternative RNA transcripts can be produced from the same genomic regionof DNA. These alternative transcripts are generally known as “variants”.More specifically, “pre-mRNA variants” are transcripts produced from thesame genomic DNA that differ from other transcripts produced from thesame genomic DNA in either their start or stop position and contain bothintronic and exonic sequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

Variants can be produced through the use of alternative signals to startor stop transcription. Pre-mRNAs and mRNAs can possess more than onestart codon or stop codon. Variants that originate from a pre-mRNA ormRNA that use alternative start codons are known as “alternative startvariants” of that pre-mRNA or mRNA. Those transcripts that use analternative stop codon are known as “alternative stop variants” of thatpre-mRNA or mRNA. One specific type of alternative stop variant is the“polyA variant” in which the multiple transcripts produced result fromthe alternative selection of one of the “polyA stop signals” by thetranscription machinery, thereby producing transcripts that terminate atunique polyA sites. Within the context of the invention, the types ofvariants described herein are also preferred target nucleic acids.

The locations on the target nucleic acid to which the preferredantisense compounds hybridize are hereinbelow referred to as “preferredtarget segments.” As used herein the term “preferred target segment” isdefined as at least an 8-nucleobase portion of a target region to whichan active antisense compound is targeted. While not wishing to be boundby theory, it is presently believed that these target segments representportions of the target nucleic acid that are accessible forhybridization.

While the specific sequences of certain preferred target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional preferred target segments may beidentified by one having ordinary skill.

Target segments 8-80 nucleobases in length comprising a stretch of atleast eight (8) consecutive nucleobases selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

Target segments can include DNA or RNA sequences that comprise at leastthe 8 consecutive nucleobases from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleobases being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 8 to about 80nucleobases). One having skill in the art armed with the preferredtarget segments illustrated herein will be able, without undueexperimentation, to identify further preferred target segments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

The oligomeric compounds are targeted to or not targeted to regions ofthe target apolipoprotein C-III nucleobase sequence (e.g., such as thosedisclosed in Examples 15 and 17) comprising nucleobases 1-50, 51-100,101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500,501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900,901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200,1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500,1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800,1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100,2101-2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400,2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700,2701-2750, 2751-2800, 2801-2850, 2851-2900, 2901-2950, 2591-3000,3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300,3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600,3601-3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900,3901-3950, 3951-3958 of SEQ ID NO: 4, or any combination thereof.

Further, the oligomeric compounds are targeted to or not targeted toregions of the target apolipoprotein C-III nucleobase sequence (e.g.,such as those disclosed in Examples 15 and 17) comprising nucleobases1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400,401-450, 451-500, 501-533 of SEQ ID NO: 18, or any combination thereof.

In one embodiment, the oligonucleotide compounds of this invention are100% complementary to these sequences or to small sequences found withineach of the above-listed sequences. Preferably, the antisense compoundscomprise at least 8 contiguous nucleobases of an antisense compounddisclosed herein. In another embodiment, the oligonucleotide compoundshave from at least 3 or 5 mismatches per 20 consecutive nucleobases inindividual nucleobase positions to these target regions. Still othercompounds of the invention are targeted to overlapping regions of theabove-identified portions of the apolipoprotein C-III sequence.

D. Screening and Target Validation

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of apolipoprotein C-III. “Modulators” are thosecompounds that decrease or increase the expression of a nucleic acidmolecule encoding apolipoprotein C-III and which comprise at least an8-nucleobase portion that is complementary to a preferred targetsegment. The screening method comprises the steps of contacting apreferred target segment of a nucleic acid molecule encodingapolipoprotein C-III with one or more candidate modulators, andselecting for one or more candidate modulators which decrease orincrease the expression of a nucleic acid molecule encodingapolipoprotein C-III. Once it is shown that the candidate modulator ormodulators are capable of modulating (e.g. either decreasing orincreasing) the expression of a nucleic acid molecule encodingapolipoprotein C-III, the modulator may then be employed in furtherinvestigative studies of the function of apolipoprotein C-III, or foruse as a research, diagnostic, or therapeutic agent in accordance withthe present invention.

The preferred target segments of the present invention may be also becombined with their respective complementary antisense compounds of thepresent invention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocessing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications (Fire et al., Nature,1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons etal., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282,430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95,15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir etal., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15,188-200). For example, such double-stranded moieties have been shown toinhibit the target by the classical hybridization of antisense strand ofthe duplex to the target, thereby triggering enzymatic degradation ofthe target (Tijsterman et al., Science, 2002, 295, 694-697).

The compounds of the present invention can also be applied in the areasof drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between apolipoprotein C-III and a disease state, phenotype,or condition. These methods include detecting or modulatingapolipoprotein C-III comprising contacting a sample, tissue, cell, ororganism with the compounds of the present invention, measuring thenucleic acid or protein level of apolipoprotein C-III and/or a relatedphenotypic or chemical endpoint at some time after treatment, andoptionally comparing the measured value to a non-treated sample orsample treated with a further compound of the invention. These methodscan also be performed in parallel or in combination with otherexperiments to determine the function of unknown genes for the processof target validation or to determine the validity of a particular geneproduct as a target for treatment or prevention of a particular disease,condition, or phenotype.

E. Kits, Research Reagents, Diagnostics, and Therapeutics

The compounds of the present invention are utilized for diagnostics,therapeutics, prophylaxis, and as research reagents and kits. In oneembodiment, such compounds of the invention are useful in areas ofobesity and metabolic-related disorders such as hyperlipidemia.Furthermore, antisense oligonucleotides, which are able to inhibit geneexpression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics, the compounds of the present invention,either alone or in combination with other compounds or therapeutics, areused as tools in differential and/or combinatorial analyses to elucidateexpression patterns of a portion or the entire complement of genesexpressed within cells and tissues.

As used herein, the term “system” is defined as any organism, cell, cellculture or tissue that expresses, or is made competent to expressproducts of the gene encoding apolipoprotein C-III. These include, butare not limited to, humans, transgenic animals, cells, cell cultures,tissues, xenografts, transplants and combinations thereof.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundsthat affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

The compounds of the invention are useful for research and diagnostics,because these compounds hybridize to nucleic acids encodingapolipoprotein C-III. For example, oligonucleotides that are shown tohybridize with such efficiency and under such conditions as disclosedherein as to be effective apolipoprotein C-III inhibitors will also beeffective primers or probes under conditions favoring gene amplificationor detection, respectively. These primers and probes are useful inmethods requiring the specific detection of nucleic acid moleculesencoding apolipoprotein C-III and in the amplification of said nucleicacid molecules for detection or for use in further studies ofapolipoprotein C-III. Hybridization of the antisense oligonucleotides,particularly the primers and probes, of the invention with a nucleicacid encoding apolipoprotein C-III can be detected by means known in theart. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabelling of the oligonucleotide or any othersuitable detection means. Kits using such detection means for detectingthe level of apolipoprotein C-III in a sample may also be prepared.

Also provided is a method of making a compound of the inventioncomprising specifically hybridizing in vitro a first nucleobase strandcomprising a sequence of at least 8 contiguous nucleobases of thesequence set forth in SEQ ID NO: 4 and/or SEQ ID NO: 18 to a secondnucleobase strand comprising a sequence sufficiently complementary tosaid first strand so as to permit stable hybridization.

The invention further provides a compound of the invention for use intherapy.

The invention further provides use of a compound or composition of theinvention in the manufacture of a medicament for the treatment of anyand all conditions disclosed herein.

Among diagnostic uses is the measurement of apolipoprotein C-III inpatients to identify those who may benefit from a treatment strategyaimed at reducing levels of apolipoprotein C-III. Such patients suitablefor diagnosis include patients with hypertriglyceridemia (e.g., todiagnose tendencies for coronary artery disease), abnormal lipidmetabolism, obesity, hyperlipidemia, among other disorders.

The specificity and sensitivity of antisense are also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat antisense compounds can be useful therapeutic modalities that canbe configured to be useful in treatment regimes for the treatment ofcells, tissues and animals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofapolipoprotein C-III is treated by administering antisense compounds inaccordance with this invention. For example, in one non-limitingembodiment, the methods comprise the step of administering to the animalin need of treatment, a therapeutically effective amount of anapolipoprotein C-III inhibitor. The apolipoprotein C-III inhibitors ofthe present invention effectively inhibit the activity of theapolipoprotein C-III protein or inhibit the expression of theapolipoprotein C-III protein. For example, such a compound that reduceslevels of apolipoprotein C-III is useful to prevent morbidity andmortality for subjects with cardiac-related disorders. For example, asdemonstrated in the examples, reduction in apolipoprotein C-III canresult in a reduction in the serum levels of cholesterol, triglycerides,and glucose. Thus, apolipoprotein C-III inhibitors are useful in thetreatment of hypertriglyceridemia, abnormal lipid metabolism, abnormalcholesterol metabolism, atherosclerosis, hyperlipidemia, diabetes,including Type 2 diabetes, obesity, cardiovascular disease, coronaryartery disease, among other disorders relating to abnormal metabolism orotherwise.

In one embodiment, the activity or expression of apolipoprotein C-III inan animal is inhibited by about 10%. Preferably, the activity orexpression of apolipoprotein C-III in an animal is inhibited by about30%. More preferably, the activity or expression of apolipoprotein C-IIIin an animal is inhibited by 50% or more. Thus, the oligomeric compoundsmodulate expression of apolipoprotein C-III mRNA by at least 10%, by atleast 20%, by at least 25%, by at least 30%, by at least 40%, by atleast 50%, by at least 60%, by at least 70%, by at least 75%, by atleast 80%, by at least 85%, by at least 90%, by at least 95%, by atleast 98%, by at least 99%, or by 100%.

For example, the reduction of the expression of apolipoprotein C-III maybe measured in serum, adipose tissue, liver or any other body fluid,tissue or organ of the animal. Preferably, the cells contained withinsaid fluids, tissues or organs being analyzed contain a nucleic acidmolecule encoding apolipoprotein C-III and/or apolipoprotein C-III.

The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

F. Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

Modified Internucleoside Linkages (Backbones)

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones containing a phosphorusatom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue, which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Modified Sugar and Internucleoside Linkages-Mimetics

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage (i.e. the backbone), of the nucleotide units arereplaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate target nucleic acid. One suchcompound, an oligonucleotide mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified Sugars

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Otherpreferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

A further preferred modification of the sugar includes Locked NucleicAcids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.The linkage is preferably a methylene (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in International Patent PublicationNos. WO 98/39352 and WO 99/14226.

Natural and Modified Nucleobases

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.and are presently preferred base substitutions, even more particularlywhen combined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

Conjugates

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992,and U.S. Pat. No. 6,287,860, the entire disclosure of which areincorporated herein by reference. Conjugate moieties include but are notlimited to lipid moieties such as a cholesterol moiety, cholic acid, athioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130 (filed Jun. 15, 1999), which is incorporated herein byreference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

Chimeric Compounds

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

The present invention also includes antisense compounds that arechimeric compounds. “Chimeric” antisense compounds or “chimeras,” in thecontext of this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, increased stability and/orincreased binding affinity for the target nucleic acid. An additionalregion of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNAse H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNAseL which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

In one embodiment, desirable chimeric oligonucleotides are 20nucleotides in length, composed of a central region consisting of ten2′-deoxynucleotides, flanked on both sides (5′ and 3′ directions) byfive 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside linkagesare phosphorothioate throughout the oligonucleotide and all cytidineresidues are 5-methylcytidines.

In another embodiment, certain preferred chimeric oligonucleotides arethose disclosed in the Examples herein. Particularly preferred chimericoligonucleotides are those referred to as ISIS 304757, ISIS 304758, ISIS304755, ISIS304800, and ISIS 304756.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

G. Formulations

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof. Accordingly, for example, the disclosure is alsodrawn to prodrugs and pharmaceutically acceptable salts of the compoundsof the invention, pharmaceutically acceptable salts of such prodrugs,and other bioequivalents. The term “prodrug” indicates a therapeuticagent that is prepared in an inactive form that is converted to anactive form (i.e., drug) within the body or cells thereof by the actionof endogenous enzymes or other chemicals and/or conditions. Inparticular, prodrug versions of the oligonucleotides of the inventionare prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivativesaccording to the methods disclosed in International Patent ApplicationPublication No. WO 93/24510 to Gosselin et al., published Dec. 9, 1993,or in International Patent Publication No. WO 94/26764 and U.S. Pat. No.5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, preferred examples of pharmaceutically acceptablesalts and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein in its entirety.

The present invention also includes pharmaceutical compositions andformulations that include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration. Pharmaceutical compositionsand formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug that may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes, which are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term that,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

In one embodiment, the present invention employs various penetrationenhancers to affect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Topical formulations are describedin detail in U.S. patent application Ser. No. 09/315,298, filed on May20, 1999, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein in its entirety. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Oralformulations for oligonucleotides and their preparation are described indetail in U.S. Published Patent Application No. 2003/0040497 (Feb. 27,2003) and its parent applications; U.S. Published Patent Application No.2003/0027780 (Feb. 6, 2003) and its parent applications; and U.S. patentapplication Ser. No. 10/071,822, filed Feb. 8, 2002, each of which isincorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents, which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited tocancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Alternatively, compositions ofthe invention may contain two or more antisense compounds targeted todifferent regions of the same nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

H. Dosing

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 μgto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and InternationalPatent Publication No. WO 02/36743; 5′-O-Dimethoxytrityl-thymidineintermediate for 5-methyl dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N-4-benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N-4-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O—[N,Ndimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2 Oligonucleotide and Oligonucleoside Synthesis

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or 5,366,878, herein incorporated by reference.

Alkylphosphonothioate oligonucleotides are prepared as described inInternational Patent Application Nos. PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, alsoidentified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 3 RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group, which has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine, which notonly cleaves the oligonucleotide from the solid support but also removesthe acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less-electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand., 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

RNA antisense compounds (RNA oligonucleotides) of the present inventioncan be synthesized by the methods herein or purchased from DharmaconResearch, Inc (Lafayette, Colo.). Once synthesized, complementary RNAantisense compounds can then be annealed by methods known in the art toform double stranded (duplexed) antisense compounds. For example,duplexes can be formed by combining 30 μl of each of the complementarystrands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90°C., then 1 hour at 37° C. The resulting duplexed antisense compounds canbe used in kits, assays, screens, or other methods to investigate therole of a target nucleic acid.

Example 4 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me]Chimeric Phosphorothioate Oligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspectrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)]ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)]chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester]ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl)phosphodiester]chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites, oxidation withiodine to generate the phosphodiester internucleotide linkages withinthe wing portions of the chimeric structures and sulfurization utilizing3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generatethe phosphorothioate internucleotide linkages for the center gap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5 Design and Screening of Duplexed Antisense Compounds TargetingApolipoprotein C-III

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense compounds of the present invention andtheir complements are designed to target apolipoprotein C-III. Thenucleobase sequence of the antisense strand of the duplex comprises atleast a portion of an oligonucleotide in Table 1. The ends of thestrands may be modified by the addition of one or more natural ormodified nucleobases to form an overhang. The sense strand of the dsRNAis then designed and synthesized as the complement of the antisensestrand and may also contain modifications or additions to eitherterminus. For example, in one embodiment, both strands of the dsRNAduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG (SEQ ID NO: 465) and having a two-nucleobaseoverhang of deoxythymidine(dT) would have the following structure(Antisense SEQ ID NO: 466, Complement SEQ ID NO: 467):

In another embodiment, a duplex comprising an antisense strand havingthe same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 465) may be preparedwith blunt ends (no single stranded overhang) as shown (Antisense SEQ IDNO: 465, Complement SEQ ID NO: 468):

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 μM. Once diluted, 30μL of each strand is combined with 15 μL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 μL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 μM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds are evaluated for theirability to modulate apolipoprotein C-III expression.

When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1™ reduced-serum medium(Gibco BRL) and then treated with 130 μL of OPTI-MEM-1™ mediumcontaining 12 μg/mL LIPOFECTIN™ reagent (Gibco BRL) and the desiredduplex antisense compound at a final concentration of 200 nM. After 5hours of treatment, the medium is replaced with fesh medium. Cells areharvested 16 hours after treatment, at which time RNA is isolated andtarget reduction measured by RT-PCR.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full-length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis were determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis 96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis 96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQapparatus) or, for individually prepared samples, on a commercial CEapparatus (e.g., Beckman P/ACE™ 5000, ABI 270 apparatus). Base andbackbone composition was confirmed by mass analysis of the compoundsutilizing electrospray-mass spectroscopy. All assay test plates werediluted from the master plate using single and multi-channel roboticpipettors. Plates were judged to be acceptable if at least 85% of thecompounds on the plate were at least 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #353872) at a density of7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville, Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

HepG2 Cells:

The human hepatoblastoma cell line HepG2 was obtained from the AmericanType Culture Collection (Manassas, Va.). HepG2 cells were routinelycultured in Eagle's MEM supplemented with 10% fetal calf serum,non-essential amino acids, and 1 mM sodium pyruvate (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Hep3B Cells:

The human hepatocellular carcinoma cell line Hep3B was obtained from theAmerican Type Culture Collection (Manassas, Va.). Hep3B cells wereroutinely cultured in Dulbeccos's MEM high glucose supplemented with 10%fetal calf serum, L-glutamine and pyridoxine hydrochloride (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 24-well plates (Falcon-Primaria #3846) at a density of50,000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Primary Mouse Hepatocytes:

Primary mouse hepatocytes were prepared from CD-1 mice purchased fromCharles River Labs (Wilmington, Mass.) and were routinely cultured inDMEM, high glucose (Invitrogen Life Technologies, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (Invitrogen Life Technologies,Carlsbad, Calif.), 100 units per ml penicillin, and 100 micrograms perml streptomycin (Invitrogen Life Technologies, Carlsbad, Calif.). Cellswere cultured to 80% confluence for use in antisense oligonucleotidetransfection experiments.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Primary Rat Hepatocytes:

Primary rat hepatocytes were prepared from Sprague-Dawley rats purchasedfrom Charles River Labs (Wilmington, Mass.) and were routinely culturedin DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (Invitrogen Life Technologies,Carlsbad, Calif.), 100 units per ml penicillin, and 100 micrograms perml streptomycin (Invitrogen Life Technologies, Carlsbad, Calif.). Cellswere cultured to 80% confluence for use in antisense oligonucleotidetransfection experiments.

Treatment with Antisense Compounds:

When cells reached 65-75% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen LifeTechnologies, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 medium containing 3.75 μg/mL LIPOFECTIN™ reagent (InvitrogenLife Technologies, Carlsbad, Calif.) and the desired concentration ofoligonucleotide. Cells are treated and data are obtained in triplicate.After 4-7 hours of treatment at 37° C., the medium was replaced withfresh medium. Cells were harvested 16-24 hours after oligonucleotidetreatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 10 Analysis of Oligonucleotide Inhibition of ApolipoproteinC-III Expression

Antisense modulation of apolipoprotein C-III expression can be assayedin a variety of ways known in the art. For example, apolipoprotein C-IIImRNA levels can be quantitated by, e.g., Northern blot analysis,competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR).Real-time quantitative PCR is presently preferred. RNA analysis can beperformed on total cellular RNA or poly(A)+mRNA. The preferred method ofRNA analysis of the present invention is the use of total cellular RNAas described in other examples herein. Methods of RNA isolation are wellknown in the art. Northern blot analysis is also routine in the art.Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISMS 7600, 7700, or 7900 Sequence DetectionSystem, available from PE-Applied Biosystems, Foster City, Calif. andused according to manufacturer's instructions.

Protein levels of apolipoprotein C-III can be quantitated in a varietyof ways well known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed toapolipoprotein C-III can be identified and obtained from a variety ofsources, such as the MSRS catalog of antibodies (Aerie Corporation,Birmingham, Mich.), or can be prepared via conventional monoclonal orpolyclonal antibody generation methods well known in the art.

Example 11 Design of Phenotypic Assays and In Vivo Studies for the Useof Apolipoprotein C-III Inhibitors

Phenotypic Assays

Once apolipoprotein C-III inhibitors have been identified by the methodsdisclosed herein, the compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of apolipoprotein C-III in health and disease.Representative phenotypic assays, which can be purchased from any one ofseveral commercial vendors, include those for determining cellviability, cytotoxicity, proliferation or cell survival (MolecularProbes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assaysincluding enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences,Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.),cell regulation, signal transduction, inflammation, oxidative processesand apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated withapolipoprotein C-III inhibitors identified from the in vitro studies aswell as control compounds at optimal concentrations which are determinedby the methods described above. At the end of the treatment period,treated and untreated cells are analyzed by one or more methods specificfor the assay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status, which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the genotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the apolipoprotein C-IIIinhibitors. Hallmark genes, or those genes suspected to be associatedwith a specific disease state, condition, or phenotype, are measured inboth treated and untreated cells.

In Vivo Studies

The individual subjects of the in vivo studies described herein arewarm-blooded vertebrate animals, which includes humans.

The clinical trial is subjected to rigorous controls to ensure thatindividuals are not unnecessarily put at risk and that they are fullyinformed about their role in the study. To account for the psychologicaleffects of receiving treatments, volunteers are randomly given placeboor apolipoprotein C-III inhibitor. Furthermore, to prevent the doctorsfrom being biased in treatments, they are not informed as to whether themedication they are administering is a apolipoprotein C-III inhibitor ora placebo. Using this randomization approach, each volunteer has thesame chance of being given either the new treatment or the placebo.

Volunteers receive either the apolipoprotein C-III inhibitor or placebofor eight week period with biological parameters associated with theindicated disease state or condition being measured at the beginning(baseline measurements before any treatment), end (after the finaltreatment), and at regular intervals during the study period. Suchmeasurements include the levels of nucleic acid molecules encodingapolipoprotein C-III or the levels of apolipoprotein C-III protein inbody fluids, tissues or organs compared to pre-treatment levels. Othermeasurements include, but are not limited to, indices of the diseasestate or condition being treated, body weight, blood pressure, serumtiters of pharmacologic indicators of disease or toxicity as well asADME (absorption, distribution, metabolism and excretion) measurements.

Information recorded for each patient includes age (years), gender,height (cm), family history of disease state or condition (yes/no),motivation rating (some/moderate/great) and number and type of previoustreatment regimens for the indicated disease or condition.

Volunteers taking part in this study are healthy adults (age 18 to 65years) and roughly an equal number of males and females participate inthe study. Volunteers with certain characteristics are equallydistributed for placebo and apolipoprotein C-III inhibitor treatment. Ingeneral, the volunteers treated with placebo have little or no responseto treatment, whereas the volunteers treated with the apolipoproteinC-III inhibitor show positive trends in their disease state or conditionindex at the conclusion of the study.

Example 12 RNA Isolation

Poly(A)+mRNA Isolation

Poly(A)+mRNA was isolated according to Miura et al., (Clin. Chem., 1996,42, 1758-1764). Other methods for poly(A)+mRNA isolation are routine inthe art. Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C., was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 150 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 150 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 1 minute. 500 μL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and incubatedfor 15 minutes and the vacuum was again applied for 1 minute. Anadditional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE wasthen added to each well of the RNEASY 96™ plate and the vacuum appliedfor a period of 90 seconds. The Buffer RPE wash was then repeated andthe vacuum was applied for an additional 3 minutes. The plate was thenremoved from the QIAVAC™ manifold and blotted dry on paper towels. Theplate was then re-attached to the QIAVAC™ manifold fitted with acollection tube rack containing 1.2 mL collection tubes. RNA was theneluted by pipetting 140 μL of RNAse free water into each well,incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN® Bio-Robot™ 9604 apparatus (Qiagen, Inc., Valencia Calif.).Essentially, after lysing of the cells on the culture plate, the plateis transferred to the robot deck where the pipetting, DNase treatmentand elution steps are carried out.

Example 13 Real-Time Quantitative PCR Analysis of Apolipoprotein C-IIImRNA Levels

Quantitation of apolipoprotein C-III mRNA levels was accomplished byreal-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.)according to manufacturer's instructions. This is a closed-tube,non-gel-based, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time. As opposed to standard PCR in which amplification productsare quantitated after the PCR is completed, products in real-timequantitative PCR are quantitated as they accumulate. This isaccomplished by including in the PCR reaction an oligonucleotide probethat anneals specifically between the forward and reverse PCR primers,and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 5′ end of the probe and a quencherdye (e.g., TAMRA, obtained from either PE-Applied Biosystems, FosterCity, Calif., Operon Technologies Inc., Alameda, Calif. or IntegratedDNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ Sequence Detection System. In each assay, aseries of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad,Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail(2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP,dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nMof probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 UnitsMuLV reverse transcriptase, and 2.5×ROX dye) to 96-well platescontaining 30 μL total RNA solution (20-200 ng). The RT reaction wascarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ reagent(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).Methods of RNA quantification by RiboGreen™ reagent are taught in Jones,L. J., et al., (Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 reader (PE Applied Biosystems) with excitation at485 nm and emission at 530 nm.

Probes and primers to human apolipoprotein C-III were designed tohybridize to a human apolipoprotein C-III sequence, using publishedsequence information (nucleotides 6238608 to 6242565 of the sequencewith GenBank accession number NT_(—)035088.1, incorporated herein as SEQID NO: 4). For human apolipoprotein C-III the PCR primers were:

forward primer: TCAGCTTCATGCAGGGTTACAT (SEQ ID NO: 5)

reverse primer: ACGCTGCTCAGTGCATCCT (SEQ ID NO: 6) and the PCR probewas: FAM-AAGCACGCCACCAAGACCGCC-TAMRA

(SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is thequencher dye. For human GAPDH the PCR primers were:

forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO: 8)

reverse primer: GAAGATGGTGATGGGATTTC GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 9)and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO:10) where JOE is the fluorescent reporter dye and TAMRA is the quencherdye.

Probes and primers to mouse apolipoprotein C-III were designed tohybridize to a mouse apolipoprotein C-III sequence, using publishedsequence information (GenBank accession number L04150.1, incorporatedherein as SEQ ID NO: 11). For mouse apolipoprotein C-III the PCR primerswere:

forward primer: TGCAGGGCTACATGGAACAA (SEQ ID NO: 12)

reverse primer: CGGACTCCTGCACGCTACTT (SEQ ID NO: 13) and the PCR probewas: FAM-CTCCAAGACGGTCCAGGATGCGC-TAMRA

(SEQ ID NO: 14) where FAM is the fluorescent reporter dye and TAMRA isthe quencher dye. For mouse GAPDH the PCR primers were:

forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 15)

reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 16) and the PCR probewas: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 17) whereJOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14 Northern Blot Analysis of Apolipoprotein C-III mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ reagent (TEL-TEST “B”Inc., Friendswood, Tex.). Total RNA was prepared followingmanufacturer's recommended protocols. Twenty micrograms of total RNA wasfractionated by electrophoresis through 1.2% agarose gels containing1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon,Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes(Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillarytransfer using a Northern/Southern Transfer buffer system (TEL-TEST “B”Inc., Friendswood, Tex.). RNA transfer was confirmed by UVvisualization. Membranes were fixed by UV cross-linking using aSTRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.)and then probed using QUICKHYB™ hybridization solution (Stratagene, LaJolla, Calif.) using manufacturer's recommendations for stringentconditions.

To detect human apolipoprotein C-III, a human apolipoprotein C-IIIspecific probe was prepared by PCR using the forward primerTCAGCTTCATGCAGGGTTACAT (SEQ ID NO: 5) and the reverse primerACGCTGCTCAGTGCATCCT (SEQ ID NO: 6). To normalize for variations inloading and transfer efficiency membranes were stripped and probed forhuman glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

To detect mouse apolipoprotein C-III, a mouse apolipoprotein C-IIIspecific probe was prepared by PCR using the forward primerTGCAGGGCTACATGGAACAA (SEQ ID NO: 12) and the reverse primerCGGACTCCTGCACGCTACTT (SEQ ID NO: 13). To normalize for variations inloading and transfer efficiency membranes were stripped and probed formouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ apparatus and IMAGEQUANT™ Software V3.3 (MolecularDynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels inuntreated controls.

Example 15 Antisense Inhibition of Human Apolipoprotein C-III Expressionby Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap

In accordance with the present invention, a series of antisensecompounds was designed to target different regions of the humanapolipoprotein C-III RNA, using published sequences (nucleotides 6238608to 6242565 of GenBank accession number NT_(—)035088.1, representing agenomic sequence, incorporated herein as SEQ ID NO: 4, and GenBankaccession number NM_(—)000040.1, incorporated herein as SEQ ID NO: 18).The compounds are shown in Table 1. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe compound binds. All compounds in Table 1 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-O-(2-methoxyethyl) nucleotides, also knownas (2′-MOE)nucleotides. The internucleoside (backbone) linkages arephosphorothioate (P═S) throughout the oligonucleotide. All cytidineresidues are 5-methylcytidines. The compounds were analyzed for theireffect on human apolipoprotein C-III mRNA levels by quantitativereal-time PCR as described in other examples herein. Data are averagesfrom three experiments in which HepG2 cells were treated with theantisense oligonucleotides of the present invention. The positivecontrol for each datapoint is identified in the table by sequence IDnumber. If present, “N.D.” indicates “no data”.

TABLE 1 Inhibition of human apolipoprotein C-III mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET SEQ CONTROL SEQ ID TARGET % ID SEQ ID ISIS * REGION NO SITESEQUENCE INHIB NO NO 167824 5′UTR 4 414 ctggagcagctgcctctagg 79 19 1167835 Coding 4 1292 ccctgcatgaagctgagaag 60 20 1 167837 Coding 18 141gtgcttcatgtaaccctgca 88 21 1 167846 Coding 4 1369 tggcctgctgggccacctgg66 22 1 167848 Coding 4 3278 tgctccagtagtctttcagg 81 23 1 167851 Coding4 3326 tgacctcagggtccaaatcc 41 24 1 304739 5′UTR 4 401ctctagggatgaactgagca 62 25 1 304740 5′UTR 4 408 cagctgcctctagggatgaa 4426 1 304741 5′UTR 18 17 ttcctggagcagctgcctct 57 27 1 304742 5′UTR 18 24acctctgttcctggagcagc 78 28 1 304743 Start Codon 18 29atggcacctctgttcctgga 78 29 1 304744 Start Codon 4 1065gggctgcatggcacctctgt 73 30 1 304745 Coding 4 1086 ggcaacaacaaggagtaccc90 31 1 304746 Coding 4 1090 ggagggcaacaacaaggagt 80 32 1 304747 Coding18 87 agctcgggcagaggccagga 49 33 1 304748 Coding 18 92tctgaagctcgggcagaggc 72 34 1 304749 Coding 18 97 cggcctctgaagctcgggca 1135 1 304750 Coding 4 1267 catcctcggcctctgaagct 49 36 1 304751 Coding 41273 gggaggcatcctcggcctct 65 37 1 304752 Coding 4 1278gagaagggaggcatcctcgg 82 38 1 304753 Coding 4 1281 gctgagaagggaggcatcct75 39 1 304754 Coding 4 1289 tgcatgaagctgagaaggga 74 40 1 304755 Coding18 143 gcgtgcttcatgtaaccctg 95 41 1 304756 Coding 4 1313ttggtggcgtgcttcatgta 92 42 1 304757 Coding 4 1328 gcatccttggcggtcttggt98 43 1 304758 Coding 4 1334 ctcagtgcatccttggcggt 97 44 1 304759 Coding4 1336 tgctcagtgcatccttggcy 93 45 1 304760 Coding 4 1347ctcctgcacgctgctcagtg 65 46 1 304761 Coding 4 1349 gactcctgcacgctgctcag77 47 1 304762 Coding 4 1358 gccacctgggactcctgcac 89 48 1 304763 Coding18 210 gcccctggcctgctgggcca 71 49 1 304764 Coding 18 211agcccctggcctgctgggcc 62 50 1 304765 Coding 4 3253 gaagccatcggtcacccagc71 51 1 304766 Coding 4 3255 ctgaagccatcggtcaccca 85 52 1 304767 Coding4 3265 tttcagggaactgaagccat 73 53 1 304768 Coding 4 3273cagtagtctttcagggaact 40 54 1 304769 Coding 4 3283 aacggtgctccagtagtctt66 55 1 304770 Coding 4 3287 ccttaacggtgctccagtag 88 56 1 304771 Coding4 3295 gaacttgtccttaacggtgc 59 57 1 304772 Coding 4 3301ctcagagaacttgtccttaa 88 58 1 304773 Coding 4 3305 agaactcagagaacttgtcc75 59 1 304774 Coding 4 3310 atcccagaactcagagaact 0 60 1 304775 Coding 43320 cagggtccaaatcccagaac 70 61 1 304776 Coding 4 3332ttggtctgacctcagggtcc 90 62 1 304777 Coding 4 3333 gttggtctgacctcagggtc84 63 1 304778 Coding 4 3339 gctgaagttggtctgacctc 81 64 1 304779 Coding4 3347 cagccacggctgaagttggt 75 65 1 304780 Stop Codon 4 3351caggcagccacggctgaagt 83 66 1 304781 Stop Codon 4 3361attgaggtctcaggcagcca 79 67 1 304782 3′UTR 4 3385 tggataggcaggtggacttg 6468 1 304783 3′UTR 18 369 ctcgcaggatggataggcag 76 69 1 304784 3′UTR 18374 aggagctcgcaggatggata 58 70 1 304785 3′UTR 18 380gacccaaggagctcgcagga 73 71 1 304786 3′UTR 18 385 tgcaggacccaaggagctcg 9272 1 304787 3′UTR 4 3417 tggagattgcaggacccaag 88 73 1 304788 3′UTR 43422 agccctggagattgcaggac 69 74 1 304789 3′UTR 4 3425ggcagccctggagattgcag 76 75 1 304790 3′UTR 4 3445 ccttttaagcaacctacagg 6576 1 304791 3′UTR 4 3450 ctgtcccttttaagcaacct 53 77 1 304792 3′UTR 43456 agaatactgtcccttttaag 72 78 1 304793 3′UTR 4 3461cactgagaatactgtccctt 67 79 1 304794 3′UTR 4 3469 taggagagcactgagaatac 5980 1 304795 3′UTR 4 3472 gggtaggagagcactgagaa 74 81 1 304796 3′UTR 43509 aggccagcatgcctggaggg 63 82 1 304797 3′UTR 4 3514ttgggaggccagcatgcctg 55 83 1 304798 3′UTR 4 3521 agctttattgggaggccagc 9084 1 304799 3′UTR 4 3526 tgtccagctttattgggagg 85 85 1 304800 3′UTR 43528 cttgtccagctttattggga 94 86 1 304801 3′UTR 4 3533agcttcttgtccagctttat 74 87 1 304802 3′UTR 4 3539 catagcagcttcttgtccag 7388 1 304803 exon:intron 4 416 acctggagcagctgcctcta 87 89 1 junction304804 exon:intron 4 424 agggcattacctggagcagc 68 90 1 junction 304805intron:exon 4 1053 acctctgttcctgcaaggaa 74 91 1 junction 304806exon:intron 4 1121 aagtgcttacgggcagaggc 78 92 1 junction 304807exon:intron 4 1380 gcgggtgtacctggcctgct 52 93 1 junction 304808 intron 42337 aaccctttgtgaactgcac 59 94 1 304809 intron 4 2405agtgagcaataccgcctgag 80 95 1 304810 intron 4 2542 cggcttgaattaggtcagg 5696 1

As shown in Table 1, SEQ ID NOs 19, 20, 21, 22, 23, 25, 27, 28, 29, 30,31, 32, 33, 34, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 55, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95 and 96 demonstrated at least 45%inhibition of human apolipoprotein C-III expression in this assay andare therefore preferred. More preferred are SEQ ID NOs 75, 86 and 85.The target regions to which these preferred sequences are complementaryare herein referred to as “preferred target segments” and are thereforepreferred for targeting by compounds of the present invention. Thesepreferred target segments are shown in Table 3. The sequences representthe reverse complement of the preferred antisense compounds shown inTable 1. “Target site” indicates the first (5′-most) nucleotide numberon the particular target nucleic acid to which the oligonucleotidebinds. Also shown in Table 3 is the species in which each of thepreferred target segments was found.

Example 16 Antisense Inhibition of Mouse Apolipoprotein C-III Expressionby Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap

In accordance with the present invention, a second series of antisensecompounds was designed to target different regions of the mouseapolipoprotein C-III RNA, using published sequences (GenBank accessionnumber L04150.1, incorporated herein as SEQ ID NO: 11). The compoundsare shown in Table 2. “Target site” indicates the first (5′-most)nucleotide number on the particular target nucleic acid to which thecompound binds. All compounds in Table 2 are chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings”. The wings arecomposed of 2′-O-(2-methoxyethyl)nucleotides, also known as(2′-MOE)nucleotides. The internucleoside (backbone) linkages arephosphorothioate (P═S) throughout the oligonucleotide. All cytidineresidues are 5-methylcytidines. The compounds were analyzed for theireffect on mouse apolipoprotein C-III mRNA levels by quantitativereal-time PCR as described in other examples herein. Data are averagesfrom three experiments in which mouse primary hepatocyte cells weretreated with the antisense oligonucleotides of the present invention. Ifpresent, “N.D.” indicates “no data”.

TABLE 2 Inhibition of mouse apolipoprotein C-III mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET SEQ ID TARGET % SEQ ID ISIS # REGION NO SITE SEQUENCE INHIB NO167858 5′UTR 11 1 tagggataaaactgagcagg 47 97 167859 5′UTR 11 21ctggagtagctagctgcttc 30 98 167860 start 11 41 gctgcatggcacctacgtac 80 99codon 167861 coding 11 62 ccacagtgaggagcgtccgg 86 100 167862 coding 1188 ggcagatgccaggagagcca 55 101 167863 coding 11 104 ctacctcttcagctcgggca56 102 167864 coding 11 121 cagcagcaaggatccctcta 83 103 167865 coding 11131 gcacagagcccagcagcaag 49 104 167867 coding 11 215ccctggccaccgcagctata 67 105 167868 coding 11 239 atctgaagtgattgtccatc 11106 167869 coding 11 254 agtagcctttcaggaatctg 57 107 167870 coding 11274 cttgtcagtaaacttgctcc 89 108 167871 coding 11 286gaagccggtgaacttgtcag 55 109 167872 coding 11 294 gaatcccagaagccggtgaa 29110 167873 coding 11 299 ggttagaatcccagaagccg 55 111 167874 coding 11319 tggagttggttggtcctcag 79 112 167875 stop 11 334 tcacgactcaatagctggag77 113 codon 167877 3′UTR 11 421 cccttaaagcaaccttcagg 71 114 1678783′UTR 11 441 agacatgagaacatactttc 81 115 167879 3′UTR 11 471catgtttaggtgagatctag 87 116 167880 3′UTR 11 496 tcttatccagctttattagg 98117

As shown in Table 2, SEQ ID NOs 97, 99, 100, 101, 102, 103, 104, 105,107, 108, 109, 111, 112, 113, 114, 115, 116 and 117 demonstrated atleast 45% inhibition of mouse apolipoprotein C-III expression in thisexperiment and are therefore preferred. More preferred are SEQ ID NOs117, 116, and 100. The target regions to which these preferred sequencesare complementary are herein referred to as “preferred target segments”and are therefore preferred for targeting by compounds of the presentinvention. These preferred target segments are shown in Table 3. Thesequences represent the reverse complement of the preferred antisensecompounds shown in Table 2. These sequences are shown to contain thymine(T) but one of skill in the art will appreciate that thymine (T) isgenerally replaced by uracil (U) in RNA sequences. “Target site”indicates the first (5′-most) nucleotide number on the particular targetnucleic acid to which the oligonucleotide binds. Also shown in Table 3is the species in which each of the preferred target segments was found.

TABLE 3 Sequence and position of preferred target segments identified inapolipoprotein C-III. TARGET REV COMP SEQ SITE SEQ ID TARGET OF SEQ IDID NO SITE SEQUENCE ID ACTIVE IN NO 82975 4 414 cctagaggcagctgctccag 19H. sapiens 118 82980 4 1292 cttctcagcttcatgcaggg 20 H. sapiens 119 8298118 141 tgcagggttacatgaagcac 21 H. sapiens 120 82985 4 1369ccaggtggcccagcaggcca 22 H. sapiens 121 82987 4 3278 cctgaaagactactggagca23 H. sapiens 122 220510 4 401 tgctcagttcatccctagag 25 H. sapiens 123220512 18 17 agaggcagctgctccaggaa 27 H. sapiens 124 220513 18 24gctgctccaggaacagaggt 28 H. sapiens 125 220514 18 29 tccaggaacagaggtgccat29 H. sapiens 126 220515 4 1065 acagaggtgccatgcagccc 30 H. sapiens 127220516 4 1086 gggtactccttgttgttgcc 31 H. sapiens 128 220517 4 1090actccttgttgttgccctcc 32 H. sapiens 129 220518 18 87 tcctggcctctgcccgagct33 H. sapiens 130 220519 18 92 gcctctgcccgagcttcaga 34 H. sapiens 131220521 4 1267 agcttcagaggccgaggatg 36 H. sapiens 132 220522 4 1273agaggccgaggatgcctccc 37 H. sapiens 133 220523 4 1278ccgaggatgcctcccttctc 38 H. sapiens 134 220524 4 1281aggatgcctcccttctcagc 39 H. sapiens 135 220525 4 1289tcccttctcagcttcatgca 40 H. sapiens 136 220526 18 143cagggttacatgaagcacgc 41 H. sapiens 137 220527 4 1313tacatgaagcacgccaccaa 42 H. sapiens 138 220528 4 1328accaagaccgccaaggatgc 43 H. sapiens 139 220529 4 1334accgccaaggatgcactgag 44 H. sapiens 140 220530 4 1336cgccaaggatgcactgagca 45 H. sapiens 141 220531 4 1347cactgagcagcgtgcaggag 46 H. sapiens 142 220532 4 1349ctgagcagcgtgcaggagtc 47 H. sapiens 143 220533 4 1358gtgcaggagtcccaggtggc 48 H. sapiens 144 220534 18 210tggcccagcaggccaggggc 49 H. sapiens 145 220535 18 211ggcccagcaggccaggggct 50 H. sapiens 146 220536 4 3253gctgggtgaccgatggcttc 51 H. sapiens 147 220537 4 3255tgggtgaccgatggcttcag 52 H. sapiens 148 220538 4 3265atggcttcagttccctgaaa 53 H. sapiens 149 220540 4 3283aagactactggagcaccgtt 55 H. sapiens 150 220541 4 3287ctactggagcaccgttaagg 56 H. sapiens 151 220542 4 3295gcaccgttaaggacaagttc 57 H. sapiens 152 220543 4 3301ttaaggacaagttctctgag 58 H. sapiens 153 220544 4 3305ggacaagttctctgagttct 59 H. sapiens 154 220546 4 3320gttctgggatttggaccctg 61 H. sapiens 155 220547 4 3332ggaccctgaggtcagaccaa 62 H. sapiens 156 220548 4 3333gaccctgaggtcagaccaac 63 H. sapiens 157 220549 4 3339gaggtcagaccaacttcagc 64 H. sapiens 158 220550 4 3347accaacttcagccgtggctg 65 H. sapiens 159 220551 4 3351acttcagccgtggctgcctg 66 H. sapiens 160 220552 4 3361tggctgcctgagacctcaat 67 H. sapiens 161 220553 4 3385caagtccacctgcctatcca 68 H. sapiens 162 220554 18 369ctgcctatccatcctgcgag 69 H. sapiens 163 220555 18 374tatccatcctgcgagctcct 70 H. sapiens 164 220556 18 380tcctgcgagctccttgggtc 71 H. sapiens 165 220557 18 385cgagctccttgggtcctgca 72 H. sapiens 166 220558 4 3417cttgggtcctgcaatctcca 73 H. sapiens 167 220559 4 3422gtcctgcaatctccagggct 74 H. sapiens 168 220560 4 3425ctgcaatctccagggctgcc 75 H. sapiens 169 220561 4 3445cctgtaggttgcttaaaagg 76 H. sapiens 170 220562 4 3450aggttgcttaaaagggacag 77 H. sapiens 171 220563 4 3456cttaaaagggacagtattct 78 H. sapiens 172 220564 4 3461aagggacagtattctcagtg 79 H. sapiens 173 220565 4 3469gtattctcagtgctctccta 80 H. sapiens 174 220566 4 3472ttctcagtgctctcctaccc 81 H. sapiens 175 220567 4 3509ccctccaggcatgctggcct 82 H. sapiens 176 220568 4 3514caggcatgctggcctcccaa 83 H. sapiens 177 220569 4 3521gctggcctcccaataaagct 84 H. sapiens 178 220570 4 3526cctcccaataaagctggaca 85 H. sapiens 179 220571 4 3528tcccaataaagctggacaag 86 H. sapiens 180 220572 4 3533ataaagctggacaagaagct 87 H. sapiens 181 220573 4 3539ctggacaagaagctgctatg 88 H. sapiens 182 220574 4 416 tagaggcagctgctccaggt89 H. sapiens 183 220575 4 424 gctgctccaggtaatgccct 90 H. sapiens 184220576 4 1053 ttccttgcaggaacagaggt 91 H. sapiens 185 220577 4 1121gcctctgcccgtaagcactt 92 H. sapiens 186 220578 4 1380agcaggccaggtacacccgc 93 H. sapiens 187 220579 4 2337gtgcagttcacaacagggtt 94 H. sapiens 188 220580 4 2405ctcaggcggtattgctcact 95 H. sapiens 189 220581 4 2542cctgacctaattcaagcccg 96 H. sapiens 190 82997 11 1 cctgctcagttttatcccta97 M. musculus 191 82999 11 41 gtacgtaggtgccatgcagc 99 M. musculus 19283000 11 62 ccggacgctcctcactgtgg 100 M. musculus 193 83001 11 88tggctctcctggcatctgcc 101 M. musculus 194 83002 11 104tgcccgagctgaagaggtag 102 M. musculus 195 83003 11 121tagagggatccttgctgctg 103 M. musculus 196 83004 11 131cttgctgctgggctctgtgc 104 M. musculus 197 83006 11 215tatagctgcggtggccaggg 105 M. musculus 198 83008 11 254cagattcctgaaaggctact 107 M. musculus 199 83009 11 274ggagcaagtttactgacaag 108 M. musculus 200 83010 11 286ctgacaagttcaccggcttc 109 M. musculus 201 83012 11 299cggcttctgggattctaacc 111 M. musculus 202 83013 11 319ctgaggaccaaccaactcca 112 M. musculus 203 83014 11 334ctccagctattgagtcgtga 113 M. musculus 204 83016 11 421cctgaaggttgctttaaggg 114 M. musculus 205 83017 11 441gaaagtatgttctcatgtct 115 M. musculus 206 83018 11 471ctagatctcacctaaacatg 116 M. musculus 207 83019 11 496cctaataaagctggataaga 117 M. musculus 208

As these “preferred target segments” have been found by experimentationto be open to, and accessible for, hybridization with the antisensecompounds of the present invention, one of skill in the art willrecognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these preferred targetsegments and consequently inhibit the expression of apolipoproteinC-III.

According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, ribozymes,external guide sequence (EGS) oligonucleotides, alternate splicers,primers, probes, and other short oligomeric compounds that hybridize toat least a portion of the target nucleic acid.

Example 17 Antisense Inhibition of Human Apolipoprotein C-III Expressionby Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap Additional Antisense Compounds

In accordance with the present invention, an additional series ofantisense compounds was designed to target different regions of thehuman apolipoprotein C-III RNA, using published sequences (nucleotides6238608 to 6242565 of the sequence with GenBank accession numberNT_(—)035088.1, representing a genomic sequence, incorporated herein asSEQ ID NO: 4, and GenBank accession number NM_(—)000040.1, incorporatedherein as SEQ ID NO: 18). The compounds are shown in Table 4. “Targetsite” indicates the first (5′-most) nucleotide number on the particulartarget sequence to which the compound binds. All compounds in Table 4are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length,composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-O-(2-methoxyethyl)nucleotides, also known as (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humanapolipoprotein C-III mRNA levels by quantitative real-time PCR asdescribed in other examples herein. Data are averages from threeexperiments in which HepG2 cells were treated with the antisenseoligonucleotides of the present invention. If present, “N.D.” indicates“no data”,

TABLE 4 Inhibition of human apolipoprotein C-III mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET SEQ SEQ ID TARGET % ID ISIS # NO SITE SEQUENCE INHIB NO 167826 41063 gctgcatggcacctctgttc 0 209 167828 4 1110 ggcagaggccaggagcgcca 0 210167830 18 91 ctgaagctcgggcagaggcc 9 211 167832 18 101tcctcggcctctgaagctcg 0 212 167840 4 1315 tcttggtggcgtgcttcatg 0 213167842 4 1335 gctcagtgcatccttggcgg 38 214 167844 4 1345cctgcacgctgctcagtgca 28 215 167847 4 3256 actgaagccatcggtcaccc 0 216167850 4 3306 cagaactcagagaacttgtc 0 217 167852 4 3336gaagttggtctgacctcagg 0 218 167853 4 3420 ccctggagattgcaggaccc 0 219167854 4 3426 gggcagccctggagattgca 22 220 167855 4 3446cccttttaagcaacctacag 27 221

Example 18 Antisense Inhibition of Human Apolipoprotein C-III Expressionby Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap Dose-Response Study in HepG2 Cells

In accordance with the present invention, a subset of the antisenseoligonucleotides from Examples 15 and 17 was further investigated in adose-response study. Treatment doses of ISIS 167842 (SEQ ID NO: 214),ISIS 167844 (SEQ ID NO: 215), ISIS 167846 (SEQ ID NO: 22), ISIS 167837(SEQ ID NO: 21), ISIS 304789 (SEQ ID NO: 75), ISIS 304799 (SEQ ID NO:85), and ISIS 304800 (SEQ ID: 86) were 50, 150 and 300 nM. The compoundswere analyzed for their effect on human apolipoprotein C-III mRNA levelsin HepG2 cells by quantitative real-time PCR as described in otherexamples herein. Data are averages from two experiments and are shown inTable 5.

TABLE 5 Inhibition of human apolipoprotein C-III mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapDose of oligonucleotide SEQ ID 50 nM 150 nM 300 nM ISIS # NO PercentInhibition 167842 214 88 77 92 167844 215 86 86 84 167846 22 79 80 79167837 21 83 86 84 304789 75 81 91 92 304799 85 82 93 88 304800 86 80 8691

These data demonstrate that the expression of apolipoprotein C-III isinhibited in a dose-dependent manner upon treatment of cells withantisense compounds targeting apolipoprotein C-III. These compounds werefurther analyzed in Hep3B cells for their ability to reduce mRNA levelsin Hep3B cells and it was determined that ISIS 167842 and 167837inhibited apolipoprotein C-III expression in a dose dependent manner inthis cell line as well.

Example 19 Antisense Inhibition Mouse Apolipoprotein C-III Expression byChimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap Dose-Response Study in Primary Mouse Hepatocytes

In accordance with the present invention, a subset of the antisenseoligonucleotides in Example 16 was further investigated in dose-responsestudies. Treatment doses with ISIS 167861 (SEQ ID NO: 100), ISIS 167870(SEQ ID NO: 108), ISIS 167879 (SEQ ID NO: 116), and ISIS 167880 (SEQ IDNO: 117) were 40, 120 and 240 nM. The compounds were analyzed for theireffect on mouse apolipoprotein C-III mRNA levels in primary hepatocytecells by quantitative real-time PCR as described in other examplesherein. Data are averages from two experiments and are shown in Table 6.

TABLE 6 Inhibition of mouse apolipoprotein C-III mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap -dose-response study Dose of oligonucleotide SEQ ID 40 nM 120 nM 240 nMISIS # NO Percent Inhibition 167861 100 48 49 61 167870 108 16 16 46167879 116 25 54 81 167880 117 76 81 93

These data demonstrate that the expression of mouse apolipoprotein C-IIIcan be inhibited in a dose-dependent manner by treatment with antisensecompounds.

Example 20 Western Blot Analysis of Apolipoprotein C-III Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to apolipoprotein C-IIIis used, with a radiolabelled or fluorescently labeled secondaryantibody directed against the primary antibody species. Bands arevisualized using a PHOSPHORIMAGER™ instrument (Molecular Dynamics,Sunnyvale Calif.).

Example 21 Effects of Antisense Inhibition of Apolipoprotein C-III (ISIS167880) on Serum Cholesterol and Triglyceride Levels

C57BL/6 mice, a strain reported to be susceptible tohyperlipidemia-induced atherosclerotic plaque formation were used in thefollowing studies to evaluate apolipoprotein C-III antisenseoligonucleotides as potential agents to lower cholesterol andtriglyceride levels.

Male C57BL/6 mice (n=8) receiving a high fat diet (60% kcal fat) wereevaluated over the course of 6 weeks for the effects of ISIS 167880 (SEQID NO: 117) on serum cholesterol and triglyceride levels. Controlanimals received saline treatment. Mice were dosed intraperitoneallyevery three days (twice a week), after fasting overnight, with 50 mg/kgISIS 167880 or saline for six weeks.

Male C57BL/6 mice fed a normal rodent diet were fasted overnight thendosed intraperitoneally every three days with saline (control), 50 mg/kgISIS 167880 (SEQ ID NO: 117) or 50 mg/kg ISIS 167879 (SEQ ID NO: 116)for two weeks.

At study termination, forty eight hours after the final injections, theanimals were sacrificed and evaluated for serum cholesterol andtriglyceride levels and compared to the saline control. Measurements ofserum cholesterol and triglyceride levels were obtained through routineclinical analysis.

High fat fed mice treated with ISIS 167880 showed a reduction in bothserum cholesterol (196 mg/dL for control animals and 137 mg/dL for ISIS167880) and triglycerides (151 mg/dL for control animals and 58 mg/dLfor ISIS 167880) by study end.

No effect was seen on serum cholesterol levels for lean mice treatedwith ISIS 167880 (91 mg/dL for control animals and 91 mg/dL for ISIS167880), however triglycerides were lowered (91 mg/dL for controlanimals and 59 mg/dL for ISIS 167880) by study end.

Lean mice treated with ISIS 167879 showed an increase in serumcholesterol (91 mg/dL for control animals and 116 mg/dL for ISIS 167879)but a reduction in triglycerides (91 mg/dL for control animals and 65mg/dL for ISIS 167879) by study end.

These results indicate that, in mice fed a high fat diet, ISIS 167880reduces cholesterol and triglyceride to levels that are comparable tolean littermates while having no deleterious effects on the leananimals. (See Table 7 for summary of in vivo data.)

Example 22 Effects of Antisense Inhibition of Apolipoprotein C-III (ISIS167880) on Serum AST and ALT Levels

C57BL/6 mice were used in the following studies to evaluate the livertoxicity of apolipoprotein C-III antisense oligonucleotides.

Male C57BL/6 mice (n=8) receiving a high fat diet (60% kcal fat) wereevaluated over the course of 6 weeks for the effects of ISIS 167880 (SEQID NO: 117) on liver enzyme (AST and ALT) levels. Control animalsreceived saline treatment. Mice were dosed intraperitoneally every threedays (twice a week), after fasting overnight, with 50 mg/kg ISIS 167880or saline for six weeks.

Male C57BL/6 mice fed a normal rodent diet were fasted overnight thendosed intraperitoneally every three days with saline (control), 50 mg/kgISIS 167880 (SEQ ID NO: 117) or 50 mg/kg ISIS 167879 (SEQ ID NO: 116)for two weeks.

At study termination and forty-eight hours after the final injections,animals were sacrificed and evaluated for serum AST and ALT levels,which were measured by routine clinical methods. Increased levels of theliver enzymes ALT and AST can indicate toxicity and liver damage.

High fat fed mice treated with ISIS 167880 showed an increase in ASTlevels over the duration of the study compared to saline controls (157IU/L for ISIS 167880, compared to 92 IU/L for saline control).

ALT levels in high fat fed mice were increased by treatments with ISIS167880 over the duration of the study compared to saline controls (64IU/L for ISIS 167880, compared to 40 IU/L for saline control).

Lean mice treated with ISIS 167880 showed no significant increase in ASTand ALT levels over the duration of the study compared to salinecontrols (AST levels of 51 IU/L for control compared to 58 IU/L for ISIS167880; ALT levels of 26 IU/L for control compared to 27 IU/L for ISIS167880).

Lean mice treated with ISIS 167879 showed no change in AST levels and adecrease in ALT levels over the duration of the study compared to salinecontrols (AST levels of 51 IU/L for control compared to 51 IU/L for ISIS167879; ALT levels of 26 IU/L for control compared to 21 IU/L for ISIS167879).

These results suggest a minor liver toxicity effect from ISIS 167880 inmice fed a high fat diet but no liver toxicity from ISIS 167880 or167879 in mice fed a normal rodent diet. (See Table 7 for summary of invivo data.)

Example 23 Effects of Antisense Inhibition of Apolipoprotein C-III (ISIS167880) on Serum Glucose Levels

Male C57BL/6 mice (n=8) receiving a high fat diet (60% kcal fat) wereevaluated over the course of 6 weeks for the effects of ISIS 167880 (SEQID NO: 117) on serum glucose levels. Control animals received salinetreatment. Mice were dosed intraperitoneally every three days (twice aweek), after fasting overnight, with 50 mg/kg ISIS 167880 or saline forsix weeks.

Male C57BL/6 mice fed a normal rodent diet were fasted overnight thendosed intraperitoneally every three days with saline (control), 50 mg/kgISIS 167880 (SEQ ID NO: 117) or 50 mg/kg ISIS 167879 (SEQ ID NO: 116)for two weeks.

At study termination and forty-eight hours after the final injections,animals were sacrificed and evaluated for serum glucose levels, whichwas measured by routine clinical methods.

In the high fat fed mice, ISIS 167880 reduced serum glucose levels to183 mg/dL, compared to the saline control of 213 mg/dL. In lean mice,ISIS 167880 had no significant effect on serum glucose levels withmeasurements of 203 mg/dL, compared to the saline control of 204 mg/dL;while ISIS 167879 only slightly increased serum glucose levels to 216mg/dL.

These results indicate that, in mice fed a high fat diet, ISIS 167880 isable to reduce serum glucose to levels comparable to lean littermates,while having no deleterious effects on the lean animals. (See Table 7for summary of in vivo data.)

Example 24 Effects of Antisense Inhibition of Apolipoprotein C-III (ISIS167880) on Apolipoprotein C-III mRNA Levels in C57BL/6 Mice

Male C57BL/6 mice received a high fat diet (60% kcal fat) fastedovernight, and dosed intraperitoneally every three days with saline or50 mg/kg ISIS 167880 (SEQ ID NO: 117) for six weeks.

Male C57BL/6 mice fed a normal rodent diet were fasted overnight thendosed intraperitoneally every three days with saline (control) or 50mg/kg ISIS 167880 (SEQ ID NO: 117) or 50 mg/kg ISIS 167879 (SEQ ID NO:116) for two weeks.

At study termination, forty-eight hours after the final injections,animals were sacrificed and evaluated for apolipoprotein C-III mRNAlevels in liver. The high fat fed mice dosed with ISIS 167880 hadapolipoprotein C-III mRNA levels 8% that of the saline treated mice. Thelean mice showed decreased apolipoprotein C-III mRNA after treatmentwith either ISIS 167880 or ISIS 167879. The lean mice dosed with ISIS167880 had apolipoprotein C-III mRNA levels 21% that of the salinetreated mice and those dosed with ISIS 167879 had apolipoprotein C-IIImRNA levels 27% that of the saline treated mice.

These results indicate that in both high fat fed mice and lean mice,antisense oligonucleotides directed against apolipoprotein C-III areable to decrease apolipoprotein C-III mRNA levels in vivo to a similarextent. (See Table 7 for summary of in vivo data.)

TABLE 7 Effects of ISIS 167880 or 167879 treatment on cholesterol,triglyceride, glucose, liver enzyme, and apolipoprotein C-III mRNA inliver, in lean and high fat fed C57BL/6 mice. Diet, Experiment durationBiological Marker Measured High Fat, Lean, units ISIS # 6 week 2 weekCholesterol control 196 91 mg/dL 167880 137 91 167879 N.D. 116Triglycerides control 151 91 mg/dL 167880  58 59 167879 N.D. 65 Glucosecontrol 213 204 mg/dL 167880 183 203 167879 N.D. 216 Liver AST control 92 51 Enzymes IU/L 167880 157 58 167879 N.D. 51 ALT control  40 26 IU/L167880  64 27 167879 N.D. 21 Apolipoprotein C-III mRNA 167880 8% 21% %of control 167879 N.D. 27%

In summary, these results indicate that, in mice fed a high fat diet,ISIS 167880 is able to reduce serum glucose, cholesterol andtriglyceride to levels comparable to lean littermates, while having nodeleterious effects on the lean animals. Furthermore, antisenseoligonucleotides directed against apolipoprotein C-III are able todecrease apolipoprotein C-III mRNA levels in vivo to a similar extent inboth high fat fed mice and lean mice. These results suggest a minorliver toxicity effect from ISIS 167880 in mice fed a high fat diet butno liver toxicity from ISIS 167880 or 167879 in mice fed a normal rodentdiet.

Example 25 Antisense Inhibition of Apolipoprotein C-III mRNA In Vivo

C57BL/6 mice, a strain reported to be susceptible tohyperlipidemia-induced atherosclerotic plaque formation, were used inthe following studies to evaluate apolipoprotein C-III antisenseoligonucleotides as potential agents to lower cholesterol andtriglyceride levels. Accordingly, in a further embodiment, C57BL/6 miceon a high-fat diet were treated with antisense oligonucleotides targetedto apolipoprotein C-III.

Male C57BL/6 mice (n=8; 7 to 8 weeks of age) receiving a high fat diet(60% kcal fat) were evaluated for apolipoprotein C-III mRNA expressionin liver after 6 weeks of treatment with antisense oligonucleotidestargeted to apolipoprotein C-III. Mice received twice weeklyintraperitoneal injections at a dose of 25 mg/kg of ISIS 167880 (SEQ IDNO: 117), ISIS 167875 (SEQ ID NO: 113), ISIS 167878 (SEQ ID NO: 115) orISIS 167879 (SEQ ID NO: 116). Control animals received saline treatmenttwice weekly for a period of 6 weeks.

At study termination, forty-eight hours after the final injections, theanimals were sacrificed and evaluated for apolipoprotein C-III mRNAexpression in liver. RNA was isolated from liver and mRNA wasquantitated as described herein. Apolipoprotein C-III mRNA levels fromeach treatment group (n=8) were averaged. Relative to saline-treatedanimals, treatment with ISIS 167875, ISIS 167878, ISIS 167879 and ISIS167880 resulted in a 24%, 56%, 50% and 77% reduction in apolipoproteinC-III mRNA levels, respectively, demonstrating that these compoundssignificantly reduced apolipoprotein C-III mRNA expression in liver.

Example 26 Effects of Antisense Inhibition of Apolipoprotein C-III onSerum Cholesterol, Triglyceride, Glucose and Serum Transaminases

In a further embodiment, the mice treated with saline or a 25 mg/kg doseof ISIS 167880 (SEQ ID NO: 117), ISIS 167875 (SEQ ID NO: 113), ISIS167878 (SEQ ID NO: 115) or ISIS 167879 (SEQ ID NO: 116) as described inExample 25 were evaluated for serum cholesterol and triglyceride levelsfollowing 6 weeks of treatment.

At study termination, forty-eight hours after the dose of saline orantisense compound, the animals were sacrificed and evaluated for serumcholesterol, triglyceride and glucose levels by routine analysis usingan Olympus Clinical Analyzer (Olympus America Inc., Melville, N.Y.). Theserum transaminases ALT and AST, increases in which can indicatehepatotoxicity, were also measured using an Olympus Clinical Analyzer(Olympus America Inc., Melville, N.Y.). The levels of serum cholesterol,triglycerides and glucose are presented in Table 8 as the average resultfrom each treatment group (n=8), in mg/dL. ALT and AST, also shown inTable 8, are also shown as the average result from each treatment group(n=8), in international units/L (IU/L).

TABLE 8 Effects of antisense inhibition of apolipoprotein C-III on serumcholesterol, triglyceride, glucose and transaminases Treatment ISIS ISISISIS ISIS Serum marker Saline 167875 167878 167879 167880 TotalCholesterol 172 197 180 132 155 mg/dL HDL Cholesterol 149 162 157 117137 mg/dL LDL Cholesterol 25 37 28 24 21 mg/dL Serum Triglyerides 126 9975 60 52 mg/dL ALT 24 555 32 45 66 IU/L AST 56 489 76 117 132 IU/LGlucose 273 234 251 189 255 mg/dL

A significant reduction in serum triglyceride levels was observedfollowing treatment with ISIS 167875, ISIS 167878, ISIS 167879 and ISIS167880, which reduced triglyercide levels 22%, 40%, 52% and 58%,respectively. This reduction in serum triglycerides correlated with thereduction in apolipoprotein C-III liver mRNA expression. Moreover,reductions in target and serum triglycerides following treatment withISIS 167878, ISIS 167879 and ISIS 167880 were not accompanied byhepatoxicity, as indicated by the lack of significant increases in ALTand AST levels. Glucose levels were significantly lowered followingtreatment with ISIS 167879.

Example 27 Effects of Antisense Inhibition of Apolipoprotein C-III onBody Weight and Organ Weight

In a further embodiment, the animals treated with saline or a 25 mg/kgdose of ISIS 167880 (SEQ ID NO: 117), ISIS 167875 (SEQ ID NO: 113), ISIS167878 (SEQ ID NO: 115) or ISIS 167879 (SEQ ID NO: 116) as described inExample 25 were evaluated for changes in body weight, fat pad, liver andspleen weights. At study termination, forty-eight hours following thefinal dose of saline or antisense compound, the animals were sacrificedand body and organ weights were measured. The data shown in Table 9represent average weights from all animals in each treatment group(n=8). Body weight is presented in grams (g), while spleen, liver andfat pad weights are presented in milligrams (mg).

TABLE 9 Effects of antisense inhibition of apolipoprotein C-III on bodyand organ weights Treatment ISIS ISIS ISIS ISIS Saline 167875 167878167879 167880 Body weight 33 30 32 28 30 (g) Liver weight 126 190 141133 146 (mg) Fat pad weight 182 125 125 61 62 (mg) Spleen weight 8 12 1212 14 (mg)

As is evident in Table 9, treatment with antisense compounds targeted tomouse apolipoprotein C-III resulted in significant reductions in fat padweight. ISIS 167875 and ISIS 167878 both led to a 31% reduction in fatpad weight, while ISIS 167879 and ISIS 167880 both resulted in a 66%lowering of fat pad weight. Body weights were not significantly changedand spleen weights were slightly increased following antisense compoundtreatment. With the exception livers from animals treated with ISIS167875, liver weights were not significantly changed.

Example 28 Effects of Antisense Inhibition of Apolipoprotein C-III onLiver Triglyceride Levels

Hepatic steatosis refers to the accumulation of lipids in the liver, or“fatty liver”, which is frequently caused by alcohol consumption,diabetes and hyperlipidemia and can progress to end-stage liver damage.Given the deleterious consequences of a fatty liver condition, it is ofuse to identify compounds that prevent or ameliorate hepatic steatosis.Hepatic steatosis is evaluated both by measurement of tissuetriglyceride content and by histologic examination of liver tissue.

In a further embodiment, liver tissue triglyceride content was assessedin the animals treated with saline or a 25 mg/kg dose of ISIS 167880(SEQ ID NO: 117), ISIS 167875 (SEQ ID NO: 113), ISIS 167878 (SEQ ID NO:115) or ISIS 167879 (SEQ ID NO: 116) as described in Example 25. Livertissue triglyceride content was measured using the Triglyceride GPOassay (Roche Diagnostics, Indianapolis, Ind.). Histological analysis wasconducted by routine procedures, whereby liver tissue was fixed inneutral-buffered formalin, embedded in paraffin, sectioned andsubsequently stained with hematoxylin and eosin, to visualize nuclei andcytoplasm, respectively. Alternatively, liver tissue was procured thenimmediately frozen, sectioned, and subsequently stained with oil red Ostain to visualize lipid deposits and counterstained with eosin to markcytoplasm. The prepared samples were evaluated by light microscopy.

Relative to saline treated mice, liver tissue triglyceride levels weresignificantly lowered, by 25%, 35%, 40% and 64% following treatment withISIS 167875, ISIS 167878, ISIS 167879 and ISIS 167880, respectively.Histological analysis of stained liver sections similarly revealed areduction in liver tissue triglycerides. Thus, as demonstrated bymeasurement of tissue triglycerides and histological analyses of livertissue sections, treatment with antisense compounds targeted toapolipoprotein C-III reduced liver triglyceride content. As such,antisense compounds targeted to apolipoprotein C-III are candidatetherapeutic agents for the prevention or amelioration of hepaticsteatosis.

Example 29 Antisense Inhibition of Apolipoprotein C-III in CynomolgusMonkey Primary Hepatocytes

In a further embodiment, antisense compounds targeted to humanapolipoprotein C-III were tested for their effects on apolipoproteinC-III expression in primary Cynomolgus monkey hepatocytes. Pre-platedprimary Cynomolgus monkey hepatocytes were purchased from InVitroTechnologies (Baltimore, Md.). Cells were cultured in high-glucose DMEM(Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10%fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.), 100units/mL and 100 μg/mL streptomycin (Invitrogen Life Technologies,Carlsbad, Calif.).

Cells at a density of 80,000 cells per well in a 24-well plate weretreated with 10, 50, 150 and 300 nM of ISIS 304789 (SEQ ID NO: 75), ISIS304799 (SEQ ID NO: 85) or ISIS 304800 (SEQ ID NO: 86). ISIS 113529(CTCTTACTGTGCTGTGGACA, SEQ ID NO: 222) served as a controloligonucleotide. ISIS 113529 is a chimeric oligonucleotide (“gapmer”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-O-(2-methoxyethyl)nucleotides, also known as (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

Following 24 hours of treatment with antisense oligonucleotides,apolipoprotein C-III mRNA was measured by real-time PCR as described byother examples herein, using the primers and probe designed to the humanapolipoprotein C-III sequence (SEQ ID NOs 5, 6 and 7) to measureCynomolgous monkey apolipoprotein C-III mRNA. Primers and probe designedto human GAPDH (SEQ ID NOs 8, 9 and 10) were used to measure Cynomolgousmonkey GAPDH mRNA expression, for the purpose of normalizing gene targetquantities obtained by real-time PCR. Untreated cells served as thecontrol to which data were normalized. Data are the average of threeexperiments and are presented in Table 10.

TABLE 10 Antisense inhibition of apolipoprotein C-III in Cynomolgusmonkey primary hepatocytes Dose of Oligonucleotide SEQ ID 10 nM 50 nM150 nM 300 nM ISIS # NO % Inhibition 304789 75 0 7 1 55 304799 85 34 6066 48 304800 86 9 53 59 57 113529 222 N.D. N.D. 0 0

Example 30 Cynomolgus Monkey Apolipoprotein C-III Sequence

In a further embodiment, a portion of the Cynomolgus monkeyapolipoprotein C-III gene was sequenced. Positions 8 to 476 of the humanapolipoprotein C-III mRNA sequence (incorporated in its entirety hereinas SEQ ID NO: 18) contain the target segment to which ISIS 304789hybridizes. The corresponding region of Cynomolgus monkey apolipoproteinC-III mRNA was sequenced. RNA was isolated and purified from primaryCynomolgus monkey hepatocytes (InVitro Technologies, Gaithersburg, Md.)and was subjected to a reverse transcriptase reaction (kit fromInvitrogen Life Technologies, Carlsbad, Calif.). The resultant cDNA wasthe substrate for 40 rounds of PCR amplification, using 5′ and 3′primers designed to positions 8 and 476, respectively, of the humanapolipoprotein C-III mRNA (Amplitaq PCR kit, Invitrogen LifeTechnologies, Carlsbad, Calif.). Following gel purification of theresultant 468 bp fragment, the forward and reverse sequencing reactionsof each product were performed by Retrogen (San Diego, Calif.). ThisCynomolgus monkey sequence is incorporated herein as SEQ ID NO: 223 andis 92% identical to positions 8 to 476 of the human apolipoprotein C-IIImRNA.

Example 31 Chimeric Phosphorothioate Oligonucleotide Having 2′-MOE Wingsand a Deoxy Gap, Targeted to Cynomolgus Monkey Apolipoprotein C-III

In a further embodiment, the sequence of Cynomolgus monkeyapolipoprotein C-III incorporated herein as SEQ ID NO: 223 was used todesign an antisense oligonucleotide having 100% complementarity toCynomolgus apolipoprotein C-III mRNA. ISIS 340340 (GGCAGCCCTGGAGGCTGCAG;incorporated herein as SEQ ID NO: 224) targets nucleotide 397 of SEQ IDNO: 223, within a region corresponding to the 3′ UTR of the humanapolipoprotein C-III to which ISIS 304789 hybridizes. ISIS 340340 is achimeric oligonucleotide (“gapmer”) 20 nucleotide in length composed ofa central “gap” region consisting of 2′deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by 5 nucleotide “wings”.The wings are composed of 2′methoxyethyl (2′-MOE) nucleotides.Internucleoside (backbone) linkages are phosphorothioate (P═S)throughout the nucleotide. All cytidine residues are 5-methyl cytidines.

Example 32 Antisense Inhibition of Rat Apolipoprotein C-III Expressionby Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap

In a further embodiment, for the purpose of designing antisenseoligonucleotides to both coding and untranslated regions of ratapolipoprotein C-III mRNA, a segment of rat apolipoprotein C-III mRNAwas sequenced to provide 3′ UTR sequence, as the published ratapolipoprotein C-III mRNA sequence is restricted to the coding region.RNA was isolated and purified from primary rat hepatocytes (InVitroTechnologies, Gaithersburg, Md.) and was subjected to a reversetranscriptase reaction (kit from Invitrogen Life Technologies, Carlsbad,Calif.). The resultant cDNA was the substrate for 40 rounds of PCRamplification (Amplitaq PCR kit, Invitrogen Life Technologies, Carlsbad,Calif.), using forward and reverse primers that anneal to the 5′-mostand 3′-most ends, respectively, of mouse apolipoprotein C-III mRNA.Following gel purification of the resultant 427 bp fragment, the forwardand reverse sequencing reactions of each product were performed byRetrogen (San Diego, Calif.). This rat sequence is incorporated hereinas SEQ ID NO: 225 and includes an additional 121 bp in the 3′ directionfrom the stop codon of apolipoprotein C-III, with respect to thepublished sequence (GenBank accession number NM_(—)012501.1,incorporated herein as SEQ ID NO: 226).

A series of antisense compounds was designed to target different regionsof the rat apolipoprotein C-III mRNA, using SEQ ID NO: 225. Thecompounds are shown in Table 11. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe compound binds. All compounds in Table 11 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-O-(2-methoxyethyl)nucleotides, also knownas (2′-MOE)nucleotides. The internucleoside (backbone) linkages arephosphorothioate (P═S) throughout the oligonucleotide. All cytidineresidues are 5-methylcytidines.

The compounds were analyzed for their effect on rat apolipoprotein C-IIImRNA levels by quantitative real-time PCR as described in other examplesherein. Probes and primers to rat apolipoprotein C-III were designed tohybridize to a rat apolipoprotein C-III sequence, using publishedsequence information (GenBank accession number NM_(—)012501.1,incorporated herein as SEQ ID NO: 226). For rat apolipoprotein C-III thePCR primers were:

forward primer: GAGGGAGAGGGATCCTTGCT (SEQ ID NO: 227)

reverse primer: GGACCGTCTTGGAGGCTTG (SEQ ID NO: 228)

and the PCR probe was: FAM-CTGGGCTCTATGCAGGGCTACATGGA-TAMRA, SEQ ID NO:229) where FAM is the fluorescent dye and TAMRA is the quencher dye. Forrat GAPDH the PCR primers were:

forward primer: TGTTCTAGAGACAGCCGCATCTT (SEQ ID NO: 230)

reverse primer: CACCGACCTTCACCATCTTGT (SEQ ID NO: 231)

and the PCR probe was JOE-TTGTGCAGTGCCAGCCTCGTCTCA-TAMRA (SEQ ID NO:232) where JOE is the fluorescent reporter dye and TAMRA is the quencherdye.

Data are from an experiment in which primary rat hepatocytes weretreated with 150 nM of the antisense oligonucleotides of the invention.Results, shown in Table 11, are expressed as percent inhibition relativeto untreated control cells. If present, “N.D.” indicates “no data”.

TABLE 11 Antisense inhibition of rat apolipoprotein C-III mRNA levels bychimeric phosphorothioate oligonucleotides having 2′-MOE wings and adeoxy gap TARGET SEQ SEQ ID TARGET % ID ISIS # REGION NO SITE SEQUENCEINHIB NO 340982 Coding 225 213 TGAACTTATCAGTGAACTTG 0 233 340987 Coding225 238 TCAGGGCCAGACTCCCAGAG 7 234 340988 Coding 225 258TTGGTGTTGTTAGTTGGTCC 0 235 340991 Coding 225 258 TTGGTGTTGTTAGTTGGTCC 0236 353932 Coding 225 10 AGAGCCACGAGGGCCACGAT 0 237 353933 Coding 225 20AGAGGCCAGGAGAGCCACGA 15 238 353934 Coding 225 30 CAGCTCGGGCAGAGGCCAGG 2239 353935 Coding 225 40 TCTCCCTCATCAGCTCGGGC 0 240 353936 Coding 225 59GCCCAGCAGCAAGGATCCCT 73 241 353937 Coding 225 69 CCTGCATAGAGCCCAGCAGC 0242 353938 Coding 225 79 TCCATGTAGCCCTGCATAGA 90 243 353940 Coding 22599 GGACCGTCTTGGAGGCTTGT 76 244 353941 Coding 225 109AGTGCATCCTGGACCGTCTT 61 245 353942 Coding 225 119 CATGCTGCTTAGTGCATCCT 0246 353943 Coding 225 129 CAGACTCCTGCATGCTGCTT 57 247 353944 Coding 225139 ACAGCTATATCAGACTCCTG 0 248 353945 Coding 225 148CTGGCCACCACAGCTATATC 0 249 353946 Coding 225 169 AAGCGATTGTCCATCCAGCC 0250 353949 Coding 225 195 TGCTCCAGTAGCCTTTCAGG 0 253 353950 Coding 225200 GAACTTGCTCCAGTAGCCTT 35 252 353951 Coding 225 204CAGTGAACTTGCTCCAGTAG 0 253 353952 Coding 225 209 CTTATCAGTGAACTTGCTCC 0254 353953 Coding 225 217 CCAGTGAACTTATCAGTGAA 0 255 353954 Coding 225222 GAGGCCAGTGAACTTATCAG 0 256 353955 Coding 225 224CCAGAGGCCAGTGAACTTAT 31 257 353956 Coding 225 229 GACTCCCAGAGGCCAGTGAA 0258 353957 Coding 225 234 GGCCAGACTCCCAGAGGCCA 0 259 353958 Coding 225247 AGTTGGTCCTCAGGGCCAGA 0 260 353959 Coding 225 250GTTAGTTGGTCCTCAGGGCC 0 261 353960 Coding 225 254 TGTTGTTAGTTGGTCCTCAG 0262 353961 Coding 225 262 AGAGTTGGTGTTGTTAGTTG 0 263 353962 Coding 225267 GCTCAAGAGTTGGTGTTGTT 0 264 353963 Coding 225 271CACGGCTCAAGAGTTGGTGT 0 265 353964 Stop 225 275 GTCTCACGGCTCAAGAGTTG 0266 Codon 353966 Stop 225 285 GAACATGGAGGTCTCACGGC 55 267 Codon 353967Stop 225 289 TCTGGAACATGGAGGTCTCA 0 268 Codon 353968 3′UTR 225 293CACATCTGGAACATGGAGGT 0 269 353969 3′UTR 225 297 CAGACACATCTGGAACATGG 0270 353970 3′UTR 225 301 TGGCCAGACACATCTGGAAC 49 271 353972 3′UTR 225309 AGGATAGATGGCCAGACACA 47 272 353973 3′UTR 225 313CAGCAGGATAGATGGCCAGA 0 273 353974 3′UTR 225 317 GAGGCAGCAGGATAGATGGC 38274 353975 3′UTR 225 321 TTCGGAGGCAGCAGGATAGA 0 275 353976 3′UTR 225 325AACCTTCGGAGGCAGCAGGA 19 276 353977 3′UTR 225 329 GAGCAACCTTCGGAGGCAGC 88277 353978 3′UTR 225 333 CTTAGAGCAACCTTCGGAGG 77 278 353979 3′UTR 225337 TCCCCTTAGAGCAACCTTCG 0 279 353980 3′UTR 225 341 ACTTTCCCCTTAGAGCAACC45 280 353981 3′UTR 225 345 ATATACTTTCCCCTTAGAGC 28 281 353982 3′UTR 225349 GAGAATATACTTTCCCCTTA 96 282 353983 3′UTR 225 353GCATGAGAATATACTTTCCC 69 283 353984 3′UTR 225 357 AAAGGCATGAGAATATACTT 47284 353985 3′UTR 225 361 GGATAAAGGCATGAGAATAT 0 285 353986 3′UTR 225 365GGAGGGATAAAGGCATGAGA 0 286 353987 3′UTR 225 386 GCATGTTTAGGTGAGGTCTG 100287 353988 3′UTR 225 390 GACAGCATGTTTAGGTGAGG 0 288 353990 3′UTR 225 398TTATTTGGGACAGCATGTTT 0 289 353991 3′UTR 225 402 GCTTTTATTTGGGACAGCAT 0290 353992 3′UTR 225 407 TCCCAGCTTTTATTTGGGAC 22 291

In a further embodiment, an additional series of oligonucleotides wasdesigned to target different regions of the rat apolipoprotein C-IIIRNA, using sequences described herein (SEQ ID NO: 225 and the sequencewith Genbank accession number NM_(—)012501.1, incorporated herein as SEQID NO: 226). The oligonucleotides are shown in Table 12. “Target site”indicates the first (5′-most) nucleotide number of the particular targetsequence to which the oligonucleotide binds. All compounds in Table 12are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length,composed of a central “gap” region consisting of eight2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by 3-nucleotide “wings.” The wings are composed of2′-O-(2-methoxyethyl)nucleotides, also known as (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

TABLE 12 Chimeric phosphorothioate oligonucleotides having 2′-MOE wingsand a deoxy gap targeted to rat apolipoprotein C-III mRNA TARGET SEQ IDTARGET SEQ ID ISIS # REGION NO SITE SEQUENCE NO 340937 Coding 226 8CACGATGAGGAGCATTCGGG 292 340938 Coding 226 13 AGGGCCACGATGAGGAGCAT 293340939 Coding 225 6 CCACGAGGGCCACGATGAGG 294 340940 Coding 225 11GAGAGCCACGAGGGCCACGA 295 340941 Coding 225 16 GCCAGGAGAGCCACGAGGGC 296340942 Coding 225 21 CAGAGGCCAGGAGAGCCACG 297 340943 Coding 225 26TCGGGCAGAGGCCAGGAGAG 298 340944 Coding 225 31 TCAGCTCGGGCAGAGGCCAG 299340945 Coding 225 36 CCTCATCAGCTCGGGCAGAG 300 340946 Coding 225 41CTCTCCCTCATCAGCTCGGG 301 340947 Coding 225 46 GATCCCTCTCCCTCATCAGC 302340948 Coding 225 51 GCAAGGATCCCTCTCCCTCA 303 340949 Coding 225 56CAGCAGCAAGGATCCCTCTC 304 340950 Coding 225 61 GAGCCCAGCAGCAAGGATCC 305340951 Coding 225 66 GCATAGAGCCCAGCAGCAAG 306 340952 Coding 225 71GCCCTGCATAGAGCCCAGCA 307 340953 Coding 225 76 ATGTAGCCCTGCATAGAGCC 308340954 Coding 225 81 GTTCCATGTAGCCCTGCATA 309 340955 Coding 225 86GGCTTGTTCCATGTAGCCCT 310 340956 Coding 225 91 TTGGAGGCTTGTTCCATGTA 311340957 Coding 225 96 CCGTCTTGGAGGCTTGTTCC 312 340958 Coding 225 101CTGGACCGTCTTGGAGGCTT 313 340959 Coding 225 106 GCATCCTGGACCGTCTTGGA 314340960 Coding 225 111 TTAGTGCATCCTGGACCGTC 315 340961 Coding 225 116GCTGCTTAGTGCATCCTGGA 316 340962 Coding 225 121 TGCATGCTGCTTAGTGCATC 317340963 Coding 225 126 ACTCCTGCATGCTGCTTAGT 318 340964 Coding 225 131ATCAGACTCCTGCATGCTGC 319 340965 Coding 225 136 GCTATATCAGACTCCTGCAT 320340966 Coding 225 141 CCACAGCTATATCAGACTCC 321 340967 Coding 225 146GGCCACCACAGCTATATCAG 322 340968 Coding 226 163 CTGCTGGCCACCACAGCTAT 323340969 Coding 226 168 AGCCCCTGCTGGCCACCACA 324 340970 Coding 226 173CATCCAGCCCCTGCTGGCCA 325 340971 Coding 226 178 TTGTCCATCCAGCCCCTGCT 326340972 Coding 226 179 ATTGTCCATCCAGCCCCTGC 327 340973 Coding 225 168AGCGATTGTCCATCCAGCCC 328 340974 Coding 225 173 TTTGAAGCGATTGTCCATCC 329340975 Coding 225 178 AGGGATTTGAAGCGATTGTC 330 340976 Coding 225 183CTTTCAGGGATTTGAAGCGA 331 340977 Coding 225 188 GTAGCCTTTCAGGGATTTGA 332340978 Coding 225 193 CTCCAGTAGCCTTTCAGGGA 333 340979 Coding 225 198ACTTGCTCCAGTAGCCTTTC 334 340980 Coding 225 203 AGTGAACTTGCTCCAGTAGC 335340981 Coding 225 208 TTATCAGTGAACTTGCTCCA 336 340983 Coding 225 218GCCAGTCAACTTATCAGTGA 337 340984 Coding 225 223 CAGAGGCCAGTGAACTTATC 338340985 Coding 225 228 ACTCCCAGAGGCCAGTGAAC 339 340986 Coding 225 233GCCAGACTCCCAGAGGCCAG 340 340989 Coding 225 248 TAGTTGGTCCTCAGGGCCAG 341340990 Coding 225 253 GTTGTTAGTTGGTCCTCAGG 342 340992 Coding 225 263AAGAGTTGGTGTTGTTAGTT 343 340993 Coding 225 268 GGCTCAAGAGTTGGTGTTGT 344340994 Stop Codon 225 272 TCACGGCTCAAGAGTTGGTG 345 353939 Coding 225 89GGAGGCTTGTTCCATGTAGC 346 353947 Coding 225 180 TCAGGGATTTGAAGCGATTG 347353948 Coding 225 190 CAGTAGCCTTTCAGGGATTT 348 353965 Stop Codon 225 281ATGGAGGTCTCACGGCTCAA 349 353971 3′ UTR 225 305 TAGATGGCCAGACACATCTG 350353989 3′ UTR 225 394 TTGGGACAGCATGTTTAGGT 351

Example 33

Antisense Inhibition of Rat Apolipoprotein C-III by ChimericPhosphorothioate Oligonucleotides Having 2′-HOE Wings and a Deoxy Gap

Dose Response Study in Primary Rat Hepatocytes

In a further embodiment, four oligonucleotides were selected foradditional dose response studies. Primary rat hepatocytes were treatedwith 10, 50, 150, and 300 nM of ISIS 167878 (SEQ ID NO: 115), ISIS167880 (SEQ ID NO: 117), ISIS 340982 (SEQ ID NO: 233), or the scrambledcontrol oligo ISIS 113529 (SEQ ID NO: 222) and mRNA levels were measured24 hours after oligonucleotide treatment as described in other examplesherein. Untreated cells served as the control to which the data werenormalized.

Results of these studies are shown in Table 13. Data are averages fromthree experiments and are expressed as percent inhibition, relative tountreated controls. Where present, “N.D.” indicates “no data”.

TABLE 13 Antisense inhibition of apolipoprotein C-III mRNA expression inprimary rat hepatocytes 24 hours after oligonucleotide treatment Dose ofoligonucleotide SEQ ID 10 nM 50 nM 150 nM 300 nM ISIS # NO % Inhibition167878 115 0 0 0 4 167880 117 21 19 20 33 340982 233 15 70 83 91 113529222 N.D. N.D. N.D. 9

As shown in Table 13, ISIS 340982 was effective at reducingapolipoprotein C-III mRNA levels in a dose-dependent manner.

Example 34 Antisense Inhibition of Rat Apolipoprotein C-III by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy GapAdditional Dose Response Study in Primary Rat Hepatocytes

In a further embodiment, an additional group of antisenseoligonucleotides targeted to rat apolipoprotein C-III was selected fordose response studies. Primary rat hepatocytes were treated with 10, 50,150 and 300 nM of ISIS 353977 (SEQ ID NO: 277), ISIS 353978 (SEQ ID NO:278), ISIS 353982 (SEQ ID NO: 282), ISIS 353983 (SEQ ID NO: 283), orISIS 353987 (SEQ ID NO: 287) for a period of 24 hours. Target expressionlevels were quantitated by real-time PCR as described herein. Untreatedcells served as the control to which data were normalized. The results,shown in Table 14, are the average of three experiments and arepresented as percent inhibition of apolipoprotein C-III mRNA, relativeto untreated control cells.

TABLE 14 Dose-dependent inhibition of apolipoprotein C-III mRNAexpression in primary rat hepatocytes 24 hours after oligonucleotidetreatment Dose of oligonucleotide SEQ ID 10 nM 50 nM 150 nM 300 nM ISIS# NO % Inhibition 353977 277 26 10 3 2 353978 278 46 23 8 5 353982 28235 21 10 2 353983 283 46 23 12 2 353987 287 38 25 12 4

These data demonstrate that ISIS 353977, ISIS 353978, ISIS 353982, ISIS353983, and ISIS 353987 effectively reduce apolipoprotein C-III mRNA ina dose-dependent manner.

Example 35 Antisense Inhibition of Rat Apolipoprotein C-III In Vivo mRNALevels

In a further embodiment, the effects of antisense inhibition ofapolipoprotein C-III in rats were evaluated. Male Sprague-Dawley rats 6weeks of age (Charles River Labs, Wilmington, Mass.) were fed a normalrodent diet. Animals received intraperitoneal injections of ISIS 340982(SEQ ID NO: 233) twice weekly for two weeks. One group of animals (n=4)received 75 mg/kg ISIS 340982 and one group of animals (n=4) received100 mg/kg ISIS 340982. Saline-treated animals (n=4) served as a controlgroup.

At the end of the treatment period, animals were sacrificed and RNA wasisolated from liver. Apolipoprotein C-III mRNA was measured as describedby other examples herein. Results from each treatment group wereaveraged and the mRNA levels in livers from ISIS 340982-treated micewere normalized to the mRNA levels in livers from saline-treated mice.Treatment with 75 mg/kg or 100 mg/kg ISIS 340982 resulted in a 69%reduction and an 84% reduction in liver apolipoprotein C-III mRNA,respectively, demonstrating that ISIS 340982 effectively inhibitedtarget mRNA expression in vivo.

Example 36 Effects of Antisense Inhibition of Rat Apolipoprotein C-IIIIn Vivo Body, Liver and Spleen Weights

In a further embodiment, the rats treated with ISIS 340782 (SEQ ID NO:233) as described in Example 35 were assessed for changes in body, liverand spleen weights. Body weights were recorded at the initiation of thestudy (Week 0). Following the two-week treatment with twice-weeklyinjections of saline or ISIS 340782 at 75 or 100 mg/kg, animals weresacrificed, forty-eight hours after the fourth and final injections, theanimals were sacrificed. Body, liver and spleen weights were recorded atstudy termination.

TABLE 15 Body, liver and spleen weights in rats treated with antisenseoligonucleotide targeted to apolipoprotein C-III Treatment with ISIS340892 Saline 75 mg/kg 100 mg/kg Week Week Week Week Week WeekMeasurement 0 2 0 2 0 2 Body weight (g) 529 536 485 448 478 425 Liverweight (g) N.D. 19 N.D. 14 N.D. 16 Spleen weight (mg) N.D. 1.1 N.D. 1.6N.D. 1.6

These data demonstrate that antisense inhibition of apolipoprotein C-IIImRNA was not associated with significant changes in body, liver orspleen weight.

Example 37 Effects of Antisense Inhibition of Rat Apolipoprotein C-IIIIn Vivo Blood Lipids and Glucose Levels

In a further embodiment, the rats treated as described in Example 35were evaluated for changes in blood total cholesterol, triglycerides,HDL-cholesterol, LDL-cholesterol, free fatty acids and glucose. Bloodsamples were collected just prior to the treatments (Week 0) andfollowing the two week treatment with twice weekly injections of salineor ISIS 340982 (SEQ ID NO: 233) at 75 or 100 mg/kg. Total cholesterol,HDL-cholesterol, LDL-cholesterol, triglyceride, free fatty acid andglucose levels were measured by routine clinical methods using anOlympus Clinical Analyzer (Olympus America Inc., Melville, N.Y.). Datafrom the four animals in each treatment group were averaged. The resultsare presented in Table 16.

TABLE 16 Effects of antisense inhibition of rat apolipoprotein C-III onblood lipids and glucose Treatment 75 mg/kg 100 mg/kg Biological SalineISIS 340982 ISIS 340982 Marker Week Week Week Week Week Week Measured 02 0 2 0 2 Triglycerides 162 162 111 24 139 17 Mg/dL Total 112 102 106 40107 31 Cholesterol Mg/dL HDL- 66 63 83 23 96 17 Cholesterol Mg/dL LDL-29 32 35 13 37 10 Cholesterol Mg/dL Free Fatty 0.48 0.46 0.72 0.70 0.570.53 Acids mEq/L Glucose 153 151 147 127 164 166 Mg/dL

From the data presented in Table 16 it is evident that ISIS 340982treatment, at both doses administered, to significantly reducedcirculating triglycerides, total cholesterol, HDL-cholesterol andLDL-cholesterol in rats. Furthermore, these animals exhibited reducedexpression of apolipoprotein C-III mRNA in liver following treatmentwith ISIS 340982.

Example 38 Effects of Antisense Inhibition of Rat Apolipoprotein C-IIIIn Vivo Serum Transaminases

In a further embodiment, the rats treated as described in Example 35were evaluated for liver toxicity following antisense oligonucleotidetreatment. Following the two week treatment with twice weekly injectionsof 75 mg/kg and 100 mg/kg ISIS 340982 (SEQ ID NO: 233), animals weresacrificed and blood was collected and processed for routine clinicalanalysis. The serum transaminases ALT and AST, increases in which canindicate hepatotoxicity, were also measured using an Olympus ClinicalAnalyzer (Olympus America Inc., Melville, N.Y.). ALT and AST levels,shown in Table 17, are shown as the average result from the 4 animals ineach treatment group, in international units/L (IU/L).

TABLE 17 Effects of treatment with ISIS 340982 on serum transaminaselevels in rats Treatment Serum 75 mg/kg 100 mg/kg Transaminase SalineISIS 340982 ISIS 340982 ALT 70 49 59 IU/L AST 93 127 147 IU/L

ALT or AST levels twice that of the saline control are consideredindicative of hepatotoxicity. These data demonstrate that ISIS 340982treatment of rats, either at a dose of 75 mg/kg or 100 mg/kg, did notresult in significant hepatotoxicity.

Example 39 Antisense Inhibition of Hamster Apolipoprotein C-IIIExpression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap

In a further embodiment, for the purpose of designing antisenseoligonucleotides to different regions of hamster apolipoprotein C-IIImRNA, a segment of Mesocricetus auratus hamster apolipoprotein C-IIImRNA was sequenced to provide a segment of coding region and 3′ UTRsequence, as no published sequence of hamster apolipoprotein C-III mRNAwas available. RNA was isolated and purified from primary hamsterhepatocytes and was subjected to a reverse transcriptase reaction (kitfrom Invitrogen Life Technologies, Carlsbad, Calif.). The resultant cDNAwas the substrate for 40 rounds of PCR amplification (Amplitaq PCR kit,Invitrogen Life Technologies, Carlsbad, Calif.) using forward andreverse primers complementary to the 5′ and 3′ ends, respectively, ofthe mouse apolipoprotein C-III mRNA sequence. Following gel purificationof the resultant 435 bp fragment, the forward and reverse sequencingreactions of each product were performed by Retrogen (San Diego,Calif.). This hamster sequence is incorporated herein as SEQ ID NO: 352.

A series of oligonucleotides was designed to target regions of thehamster apolipoprotein C-III mRNA (SEQ ID NO: 352). The oligonucleotidesare shown in Table 18. “Target site” indicates the first (5′-most)nucleotide number on the particular target sequence to which theoligonucleotide binds. All compounds in Table 18 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.”The wings are composed of 2′-O-(2-methoxyethyl)nucleotides, also knownas (2′-MOE)nucleotides. The internucleoside (backbone) linkages arephosphorothioate (P═S) throughout the oligonucleotide. All cytidineresidues are 5-methylcytidines.

The compounds were analyzed for their effect on hamster apolipoproteinC-III levels in primary hamster hepatocytes by quantitative real-timePCR as described in other examples herein. Probes and primers to hamsterapolipoprotein C-III were designed to hybridize to a hamsterapolipoprotein C-III sequence, using the hamster mRNA sequence describedherein (SEQ ID NO: 352). For hamster apolipoprotein CIII the PCR primerswere:

forward primer: CGCTAACCAGCATGCAAAAG (SEQ ID NO: 353)

reverse primer: CACCGTCCATCCAGTCCC(SEQ ID NO: 354) and the

PCR probe was: FAM-CTGAGGTGGCTGTGCGGGCC-TAMRA (SEQ ID NO: 355) where FAMis the fluorescent dye and TAMRA is the quencher dye.

For hamster GAPDH the PCR primers were:

forward primer: CCAGCCTCGCTCCGG (SEQ ID NO: 356)

reverse primer: CCAATACGGCCAAATCCG (SEQ ID NO: 357)

and the PCR probe was JOE-ACGCAATGGTGAAGGTCGGCG-TAMRA (SEQ ID NO: 358)where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Data are from an experiment in which primary hamster hepatocytes weretreated with 150 nM of the oligonucleotides of the present invention.The data, shown in Table 18, are normalized to untreated control cells.If present, “N.D.” indicates “no data.”

TABLE 18 Antisense inhibition of hamster apolipoprotein C-III mRNAlevels by chimeric phosphorothioate oligonucleotides having 2′-MOE wingsand a deoxy gap TARGET SEQ SEQ ID TARGET % ID ISIS # REGION NO SITESEQUENCE INHIB NO 352929 Coding 352 5 TGCCAAGAGGGCAACAATAG 17 359 352930Coding 352 10 AGGAGTGCCAAGAGGGCAAC 62 360 352931 Coding 352 16GATGCCAGGAGTGCCAAGAG 50 361 352932 Coding 352 20 GGCAGATGCCAGGAGTGCCA 51362 352933 Coding 352 39 CTCTACCTCATTAGCTTCGG 0 363 352934 Coding 352 41CCCTCTACCTCATTAGCTTC 47 364 352935 Coding 352 44 GACCCCTCTACCTCATTAGC 0365 352936 Coding 352 49 GCAAGGACCCCTCTACCTCA 15 366 352937 Coding 35254 CAGCAGCAAGGACCCCTCTA 45 367 352938 Coding 352 59 GAGCCCAGCAGCAAGGACCC0 368 352939 Coding 352 65 TGCACAGAGCCCAGCAGCAA 84 369 352940 Coding 35270 AGCCCTGCACAGAGCCCAGC 0 370 352941 Coding 352 75 CATGTAGCCCTGCACAGAGC0 371 352942 Coding 352 80 TGTTCCATGTAGCCCTGCAC 49 372 352943 Coding 35285 TGGCCTGTTCCATGTAGCCC 55 373 352945 Coding 352 95 ACCTTCTTGGTGGCCTGTTC62 374 352946 Coding 352 106 GCGCATCCTGGACCTTCTTG 0 375 352948 Coding352 115 TGCTGGTTAGCGCATCCTGG 0 376 352949 Coding 352 120TTGCATGCTGGTTAGCGCAT 3 377 352950 Coding 352 125 GACTTTTGCATGCTGGTTAG 59378 352951 Coding 352 130 CCTCAGACTTTTGCATGCTG 72 379 352952 Coding 352135 AGCCACCTCAGACTTTTGCA 75 380 352953 Coding 352 140CGCACAGCCACCTCAGACTT 64 381 352955 Coding 352 153 CCAGTCCCTGGCCCGCACAG66 382 352956 Coding 352 159 GTCCATCCAGTCCCTGGCCC 73 383 352957 Coding352 161 CCGTCCATCCAGTCCCTGGC 0 384 352958 Coding 352 165GCCACCGTCCATCCAGTCCC 0 385 352959 Coding 352 170 GTGAAGCCACCGTCCATCCA 12386 352960 Coding 352 174 GGAGGTGAAGCCACCGTCCA 0 387 352961 Coding 352193 TGCTCCAGTAGCTTTTCAGG 59 388 352962 Coding 352 200GTAAATGTGCTCCAGTAGCT 66 389 352963 Coding 352 205 TGTCAGTAAATGTGCTCCAG78 390 352965 Coding 352 214 TGGAGACCGTGTCAGTAAAT 38 391 352966 Coding352 217 GGCTGGAGACCGTGTCAGTA 66 392 352967 Coding 352 221CAGAGGCTGGAGACCGTGTC 13 393 352968 Coding 352 225 ATCCCAGAGGCTGGAGACCG 0394 352969 Coding 352 230 GAAGAATCCCAGAGGCTGGA 54 395 352970 Coding 352269 TCTCAAGGCTCAGTAGCTGG 0 396 352971 Coding 352 275TAGAGGTCTCAAGGCTCAGT 70 397 352972 Stop Codon 352 280GAACGTAGAGGTCTCAAGGC 61 398 352973 Stop Codon 352 286CATTTGGAACGTAGAGGTCT 64 399 352974 3′ UTR 352 292 CAAGCACATTTGGAACGTAG 0400 352975 3′ UTR 352 300 TGGACACACAAGCACATTTG 0 401 352976 3′ UTR 352305 CAGGATGGACACACAAGCAC 43 402 352977 3′ UTR 352 311GGCCAGCAGGATGGACACAC 81 403 352978 3′ UTR 352 318 GCCCAGAGGCCAGCAGGATG60 404 352979 3′ UTR 352 348 CCTTTCAAACAACCTTCAGG 56 405 352980 3′ UTR352 402 GGACAGCATGTTTAGGTGAC 67 406

In a further embodiment, an additional series of oligonucleotides wasdesigned to target different regions of the hamster apolipoprotein C-IIIRNA described herein (SEQ ID NO: 352). The oligonucleotides are shown inTable 19. “Target site” indicates the first (5′-most) nucleotide numberon the particular target sequence to which the oligonucleotide binds.All compounds in Table 19 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting ofeight 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by 3-nucleotide “wings.” The wings are composed of2′-O-(2-methoxyethyl)nucleotides, also known as (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

TABLE 19 Chimeric phosphorothioate oligonucleotides having 2′-MOE wingsand a deoxy gap targeted to hamster apolipoprotein C-III mRNA TARGET SEQSEQ ID TARGET ID ISIS # REGION NO SITE SEQUENCE NO 352944 Coding 352 90CTTGGTGGCCTGTTCCATGT 407 352947 Coding 352 110 GTTAGCGCATCCTGGACCTT 408352954 Coding 352 145 TGGCCCGCACAGCCACCTCA 409 352964 Coding 352 210GACCGTGTCAGTAAATGTGC 410 356295 Coding 352 1 AAGAGGGCAACAATAGGAGT 412356296 Coding 352 6 GTGCCAAGAGGGCAACAATA 412 356297 Coding 352 15ATGCCAGGAGTGCCAAGAGG 413 356298 Coding 352 25 CTTCGGGCAGATGCCAGGAG 414356299 Coding 352 31 CATTAGCTTCGGGCAGATGC 415 356300 Coding 352 60AGAGCCCAGCAGCAAGGACC 416 356301 Coding 352 86 GTGGCCTGTTCCATGTAGCC 417356302 Coding 352 91 TCTTGGTGGCCTGTTCCATG 418 356303 Coding 352 96GACCTTCTTGGTGGCCTGTT 419 356304 Coding 352 101 TCCTGGACCTTCTTGGTGGC 420356305 Coding 352 111 GGTTAGCGCATCCTGGACCT 421 356306 Coding 352 116ATGCTGGTTAGCGCATCCTG 422 356307 Coding 352 121 TTTGCATGCTGGTTAGCGCA 423356308 Coding 352 126 AGACTTTTGCATGCTGGTTA 424 356309 Coding 352 131ACCTCAGACTTTTGCATGCT 425 356310 Coding 352 136 CAGCCACCTCAGACTTTTGC 426356311 Coding 352 141 CCGCACAGCCACCTCAGACT 427 356312 Coding 352 146CTGGCCCGCACAGCCACCTC 428 356313 Coding 352 151 AGTCCCTGGCCCGCACAGCC 429356314 Coding 352 156 CATCCAGTCCCTGGCCCGCA 430 356315 Coding 352 166AGCCACCGTCCATCCAGTCC 431 356316 Coding 352 171 GGTGAAGCCACCGTCCATCC 432356317 Coding 352 176 AGGGAGGTGAAGCCACCGTC 433 356318 Coding 352 181TTTTCAGGGAGGTGAAGCCA 434 356319 Coding 352 187 AGTAGCTTTTCAGGGAGGTG 435356320 Coding 352 198 AAATGTGCTCCAGTAGCTTT 436 356321 Coding 352 203TCAGTAAATGTGCTCCAGTA 437 356322 Coding 352 208 CCGTGTCAGTAAATGTGCTC 438356323 Coding 352 213 GGAGACCGTGTCAGTAAATG 439 356324 Coding 352 218AGGCTGGAGACCGTGTCAGT 440 356325 Coding 352 223 CCCAGAGGCTGGAGACCGTG 441356326 Coding 352 228 AGAATCCCAGAGGCTGGAGA 442 356327 Stop Codon 352 274AGAGGTCTCAAGGCTCAGTA 443 356328 Stop Codon 352 279 AACGTAGAGGTCTCAAGGCT444 356329 Stop Codon 352 284 TTTGGAACGTAGAGGTCTCA 445 356330 3′ UTR 352289 GCACATTTGGAACGTAGAGG 446 356331 3′ UTR 352 294 CACAAGCACATTTGGAACGT447 356332 3′ UTR 352 299 GGACACACAAGCACATTTGG 448 356333 3′ UTR 352 304AGGATGGACACACAAGCACA 449 356334 3′ UTR 352 309 CCAGCAGGATGGACACACAA 450356335 3′ UTR 352 314 AGAGGCCAGCAGGATGGACA 451 356336 3′ UTR 352 319GGCCCAGAGGCCAGCAGGAT 452 356337 3′ UTR 352 324 ACCCAGGCCCAGAGGCCAGC 453356338 3′ UTR 352 329 GGGCCACCCAGGCCCAGAGG 454 356339 3′ UTR 352 353CTTTCCCTTTCAAACAACCT 455 356340 3′ UTR 352 358 CAATACTTICCCTTTCAAAC 456356341 3′ UTR 352 363 CATGACAATACTTTCCCTTT 457 356342 3′ UTR 352 368GAAAACATGACAATACTTTC 458 356343 3′ UTR 352 373 GGGATGAAAACATGACAATA 459356344 3′ UTR 352 396 CATGTTTAGGTGACTTCTGG 460 356345 3′ UTR 352 401GACAGCATGTTTAGGTGACT 461 356346 3′ UTR 352 406 TTTAGGACAGCATGTTTAGG 462356347 3′ UTR 352 411 CTTTATTTAGGACAGCATGT 463 356348 3′ UTR 352 416TCCAGCTTTATTTAGGACAG 464

Example 40 Antisense Inhibition of Hamster Apolipoprotein C-III byChimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap Dose Response Studies in Primary Hamster Hepatocytes

In a further embodiment, six oligonucleotides targeted to hamsterapolipoprotein C-III were selected for additional dose response studies.Primary hamster hepatocytes were treated with 50, 150, and 300 nM ofISIS 352939 (SEQ ID NO: 369), ISIS 352952 (SEQ ID NO: 380), ISIS 352962(SEQ ID NO: 389), ISIS 352963 (SEQ ID NO: 390), ISIS 352971 (SEQ ID NO:397), or ISIS 352977 (SEQ ID NO: 403) and mRNA levels were measured 24hours after oligonucleotide treatment as described in other examplesherein. Untreated cells served as the control to which the data werenormalized.

Results of these studies are shown in Table 20. Data are averages fromthree experiments and are expressed as percent inhibition, relative tountreated controls.

TABLE 20 Inhibition of apolipoprotein C-III mRNA expression in primaryhamster hepatocytes 24 hours after oligonucleotide treatment Dose ofoligonucleotide SEQ ID 50 nM 150 nM 300 nM ISIS # NO % Inhibition 352939369 46 64 82 352952 380 59 68 60 352962 389 84 0 22 352963 390 0 0 42352971 397 0 27 0 352977 403 48 72 56

As shown in Table 20, ISIS 352939 was effective at reducing hamsterapolipoprotein C-III mRNA levels in a dose-dependent manner.

Example 41 Antisense Oligonucleotides Targeted to Mouse ApolipoproteinC-III

In a further embodiment, additional antisense oligonucleotides targetingmouse apolipoprotein C-III were designed using published sequenceinformation (GenBank accession number L04150.1, incorporated herein asSEQ ID NO: 11). Both target nucleotide position 496 of SEQ ID NO: 11, asdoes ISIS 167880 (SEQ ID NO: 117), but vary in chemical compositionrelative to ISIS 167880. ISIS 340995 is 20 nucleotides in length,composed of a central gap region 10 nucleotides in length, wherein thegap contains both 2′ deoxynucleotides and 2′-MOE (MOE)nucleotides. Thenucleotide composition is shown in Table 21, where 2′-MOE nucleotidesare indicated in bold type, and 2′ deoxynucleotides are underscored. Thegap is flanked on both sides (5′ and 3′ ends) by 5 nucleotide “wings”composed of 2′-MOE nucleotides. ISIS 340997 (SEQ ID NO: 117) is 20nucleotides in length and uniformly composed of 2′-MOE nucleotides.Throughout both ISIS 340995 and ISIS 340997, internucleoside (backbone)linkages are phosphorothioate and all cytidines residues are unmodifiedcytidines.

TABLE 21 Antisense oligonucleotides targeted to mouse apolipoproteinC-III Target ISIS SEQ ID Target SEQ ID NO Region NO Site SEQUENCE NO340995 3′ UTR 11 496 TCTTA TC C AG C TT T A TTAGG 117 340997 3′ UTR 11496 TCTTATCCAGCTTTATTAGG 117

1. A compound comprising a modified oligonucleotide consisting of 12 to30 linked nucleosides comprising an at least 8 consecutive nucleobaseportion complementary to an equal number of nucleobases of nucleotides3533-3552 of SEQ ID NO: 4, wherein said modified oligonucleotide is atleast 90% complementary to SEQ ID NO:
 4. 2. The compound of claim 1,consisting of a single-stranded modified oligonucleotide.
 3. Thecompound of claim 1, wherein said modified oligonucleotide is at least95% complementary to SEQ ID NO:
 4. 4. The compound of claim 1, whereinsaid modified oligonucleotide is 100% complementary to SEQ ID NO:
 4. 5.The compound of claim 2, wherein at least one internucleoside linkage ofsaid modified oligonucleotide is a modified internucleoside linkage. 6.The compound of claim 5, wherein each internucleoside linkage is aphosphorothioate internucleoside linkage.
 7. The compound of claim 2,wherein at least one nucleoside comprises a modified sugar.
 8. Thecompound of claim 7, wherein at least one modified sugar is a bicyclicsugar.
 9. The compound of claim 7, wherein at least one modified sugarcomprises a 2′-O-methoxyethyl.
 10. The compound of claim 1, wherein themodified oligonucleotide comprises: a gap segment consisting of linkeddeoxynucleosides; a 5′ wing segment consisting of linked nucleosides; a3′ wing segment consisting of linked nucleosides; wherein the gapsegment is positioned between the 5′ wing segment and the 3′ wingsegment and wherein each nucleoside of each wing segment comprises amodified sugar.
 11. The compound of claim 10, wherein the modifiedoligonucleotide comprises: a gap segment consisting of ten linkeddeoxynucleosides; a 5′ wing segment consisting of five linkednucleosides; a 3′ wing segment consisting of five linked nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar; wherein each cytosine residue ofthe modified oligonucleotide is a 5-methylcytosine, and wherein eachinternucleoside linkage of said modified oligonucleotide is aphosphorothioate linkage.
 12. The compound of claim 11, wherein themodified oligonucleotide consists of 20 linked nucleosides.
 13. Thecompound of claim 12, wherein the 20 linked nucleosides arecomplementary to nucleotides 3533-3552 of SEQ ID NO:
 4. 14. The compoundof claim 1, wherein said modified oligonucleotide consists of thenucleobase sequence of SEQ ID NO:
 87. 15. The compound of claim 1,wherein said modified oligonucleotide consists of the nucleobasesequence of SEQ ID NO: 87 and comprises: a gap segment consisting of tenlinked deoxynucleosides; a 5′ wing segment consisting of five linkednucleosides; a 3′ wing segment consisting of five linked nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar, wherein each internucleosidelinkage of said modified oligonucleotide is a phosphorothioate linkage,and wherein each cytosine residue of said modified oligonucleotide is a5-methylcytosine.
 16. A composition comprising the compound of claim 1or a pharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier or diluent.
 17. The composition of claim 16, whereinsaid modified oligonucleotide consists of the nucleobase sequence of SEQID NO: 87 and comprises: a gap segment consisting of ten linkeddeoxynucleosides; a 5′ wing segment consisting of five linkednucleosides; a 3′ wing segment consisting of five linked nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar, wherein each internucleosidelinkage of said modified oligonucleotide is a phosphorothioate linkage,and wherein each cytosine residue of said modified oligonucleotide is a5-methylcytosine.
 18. A modified oligonucleotide consisting of 20 linkednucleosides comprising the nucleobase sequence of SEQ ID NO: 87 and: agap segment consisting of ten linked deoxynucleosides; a 5′ wing segmentconsisting of five linked nucleosides; a 3′ wing segment consisting offive linked nucleosides; wherein the gap segment is positioned betweenthe 5′ wing segment and the 3′ wing segment, wherein each nucleoside ofeach wing segment comprises a 2′-O-methoxyethyl sugar, wherein eachinternucleoside linkage of said modified oligonucleotide is aphosphorothioate linkage, and wherein each cytosine residue of saidmodified oligonucleotide is a 5-methylcytosine.